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Repression of human papillomavirus (HPV) E6 and E7 oncogenes in established cervical carcinoma cell lines causes senescence due to reactivation of cellular tumor suppressor pathways. Here, we determined whether ongoing expression of HPV16 or HPV18 oncogenes is required for the proliferation of primary human cervical carcinoma cells in serum-free conditions at low passage number after isolation from patients. We used an SV40 viral vector expressing the bovine papillomavirus E2 protein to repress E6 and E7 in these cells. To enable efficient SV40 infection and E2 gene delivery, we first incubated the primary cervical cancer cells with the ganglioside GM1, a cell-surface receptor for SV40 limiting in these cells. Repression of HPV in primary cervical carcinoma cells caused them to undergo senescence, but the E2 protein had little effect on HPV-negative primary cells. These data suggest that E6 and E7 dependence is an inherent property of human cervical cancer cells.
Cervical carcinoma is initiated by infection with high-risk human papillomaviruses (HPV) such as HPV16 or 18 (Bosch et al., 2002). Expression of the HPV E6 and E7 oncogenes can immortalize primary, cultured human keratinocytes, the cells that give rise to cervical carcinomas (Durst et al., 1987; Hawley-Nelson et al., 1989; Hudson et al., 1990; Munger et al., 1989; Pirisi et al., 1987), and estrogen-treated transgenic mice expressing HPV16 E6 and E7 develop cervical carcinomas (Arbeit, Howley, and Hanahan, 1996). High-risk HPV E6 binds to p53, a tumor suppressor, and stimulates its ubiquitin-mediated degradation (Howie, Katzenellenbogen, and Galloway, 2009). High-risk HPV E7 binds to the p105Rb and other members of the retinoblastoma (Rb) family, causing their dissociation from E2F proteins and degradation of the active hypophosphorylated form of p105Rb, releasing E2F to activate genes required for cell cycle progression (Moody and Laimins, 2010). Transcription of E6 and E7 can be repressed by the papillomavirus E2 protein, which binds to the HPV E6/E7 promoter in the long control region of the viral genome (Thierry and Yaniv, 1987). Viral DNA integration or mutations that interfere with expression or activity of the E2 protein frequently occur during cervical carcinogenesis, leading to elevated expression of E6 and E7 (May et al., 1994; Schneider-Maunoury, Croissant, and Orth, 1987), which are continuously expressed in cervical carcinoma cell lines (Baker et al., 1987; Schwarz et al., 1985).
The proliferation and survival of cervical cancer cell lines requires ongoing expression of E6 and E7. Introduction of the HPV or bovine papillomavirus (BPV) E2 gene or siRNAs against HPV E6 and E7 inhibits the proliferation of cervical carcinoma cell lines or causes apoptosis (Desaintes et al., 1997; Dowhanick, McBride, and Howley, 1995; Hwang et al., 1993; Jiang and Milner, 2002; McBride, Romanczuk, and Howley, 1991; Parish et al., 2006). Repression of HPV18 E6 and E7 by BPV-E2 in HeLa cervical cancer cells results in growth arrest and cellular senescence due to reactivation of the p53 and Rb tumor suppressor pathways (Goodwin and DiMaio, 2000; Goodwin et al., 2000; Wells et al., 2000). Similarly, in a transgenic mouse model, repression of E7 expression driven by a tetracycline-regulated promoter leads to regression of high-grade cervical dysplasia and tumors (Jabbar et al., 2009). However, murine keratinocytes differ in some regards from their human counterparts (Balmain and Harris, 2000; Chaturvedi et al., 2004; Dotto, 1998), and cervical carcinogenesis in mice differs in certain aspects from human carcinogenesis. For example, the latency period of tumor formation in these mice is measured in months, whereas decades typically pass in women between HPV infection and the development of invasive cancer. It is thought that additional cellular mutations accumulate in infected human cells during this protracted latency period. Nevertheless, the fact that human cervical carcinoma cell lines and tumors in transgenic mice depend on HPV oncogene expression suggests that the E6 and E7 genes are potential targets for treatment of human cervical carcinoma.
It is not known whether dependence on E6 and E7 is an inherent property of human cervical carcinoma cells or whether this trait is acquired during the establishment or propagation of continuous cell lines. Continuous cell lines can be established from only a minority of primary tumors, and several pieces of evidence indicate that established cancer cell lines are not necessarily representative of the tumor of origin (Bignotti et al., 2006; Dairkee et al., 2004; Ertel et al., 2006; Lee et al., 2006; Santin et al., 2004; van Staveren et al., 2007; van Staveren et al., 2009). For example, analysis of gene expression profiles of the 60 cancer cells lines in the NC60 panel revealed only a 5 percent overlap in up-regulated genes compared to their corresponding tumor tissue (Sandberg and Ernberg, 2005). In addition, a comprehensive microarray-based analysis of cervical cancers and cervical cancer cell lines demonstrated that cervical cancers were more similar to normal primary cervical epithelial cells than they were to cervical cancer cell lines, indicating that the transcriptional changes that occur during the transition of normal cells to cancer cells are less pronounced than the changes that occur during the conversion of cancer cells into established cell lines (Carlson, Iyer, and Marcotte, 2007). These problems are a particular concern for cervical cancer cell lines, most of which have been in culture for decades (60 years in the case of HeLa cells). Thus, if a small population of cells that were dependent upon E6 and E7 possessed even a slight growth advantage in vitro, or if E6/E7-dependent cells were more likely to accumulate mutations that conferred a growth advantage during in vitro culture, these cells would eventually overgrow the culture, giving rise to a cell line that has diverged from its tumor of origin in terms of its oncogene dependence. In fact, HPV E6 and E7 engender a state of genomic instability and increased mutagenesis, which provides an accelerated path for cell lines to diverge from the original cancer (Moody and Laimins, 2010).
It might be expected that cells transformed by HPV in infected women would initially depend on the E6 and E7 oncogenes, and that the accumulation of mutations as the lesions progress in vivo or the cells are passaged in culture is likely to gradually render the cells independent of growth regulatory signals, so that they lose a requirement for E6 and E7. On the other hand, it is also possible that cancer cells do not require a particular oncogene for continued proliferation in culture until they are passaged in vitro. For example, in studies of a lung cancer cell line dependent on a mutant epidermal growth factor receptor, Turke et al. showed that in vitro culture of these cells in the presence of hepatocyte growth factor can generate cells that require continued signaling by c-Met, the hepatocyte growth factor receptor, as well as the EGF receptor (Turke et al., 2010). Published reports suggest a mechanism by which HPV E6/E7 could confer a growth advantage to cervical cancer cell lines cultured in vitro. Cervical carcinoma cell lines are typically maintained in high concentrations of calcium and fetal bovine serum (FBS). These conditions induce terminal differentiation of primary human keratinocytes and growth arrest. However, high-risk HPV E6 and E7 expression inhibits terminal differentiation of primary human keratinocytes and allows a small fraction of cells to proliferate in the presence of calcium and FBS (Munger et al., 1989; Pei et al., 1998; Schlegel et al., 1988; Sherman and Schlegel, 1996). Therefore, the standard conditions used to culture cell lines may have selected for expression of E6 and E7 to prevent terminal differentiation. If this were the case, the demonstrated requirement for E6/E7 in cell lines might reflect the ongoing need of these genes to continuously block terminal differentiation in response to serum and calcium in vitro, rather than an intrinsic growth property of the cells.
To minimize the risk of selection or genetic drift in testing the requirement for E6 and E7 in cervical cancer cells, we decided to assess the requirement for E6 and E7 in primary cultures of human cervical carcinoma cells. In work published by us and by others, gene expression profiling showed that various types of unmanipulated tumors are more similar to low passage primary cancer cells than they are to established cell lines derived from the same tumors (Bignotti et al., 2006; Dairkee et al., 2004; Lee et al., 2006; Santin et al., 2004). Thus, not surprisingly, primary cervical cancer cells are more representative of the original tumors than are long-term, established cell lines. There is a previous report that expression of BPV-E2 inhibited DNA synthesis in three early passage cervical carcinoma cell lines containing HPV16 DNA (Moon et al., 2001), but these cells were established and maintained in high calcium and FBS, so may have been subjected to selective pressure for E6/E7 dependence as described above.
In the experiments reported here, we isolated primary cancer cells from tumor biopsies of four patients with cervical carcinoma and cultured them for a minimal number of passages in low concentrations of calcium and in the absence of serum to prevent terminal differentiation and in vitro selection for E6/E7 dependence. For these experiments, we wished to use an SV40 recombinant virus vector to express the BPV-E2 protein, because this is an extremely efficient system to repress E6 and E7 in HeLa cells. However, most cells we tested infected poorly with this vector. By exploiting our understanding of factors that dictate SV40 infection, we were able to markedly enhance infection by the SV40 vector by treating the cells with the ganglioside GM1, a cellular receptor of SV40 (Tsai et al., 2003), prior to infection. GM1 added to the culture medium is incorporated into the plasma membrane (Schwarzmann, 2001), thus increasing the cell surface GM1 that can be used by SV40 for infection. By using this approach, we demonstrated that primary cervical carcinoma cells are indeed dependent on E6 and E7 for continued growth.
Primary cervical carcinoma cells were isolated from surgical biopsies as described previously (Santin et al., 2005) and cultured in serum-free keratinocyte medium, which contains low concentrations of calcium to prevent differentiation. Characteristics of these cells including the resident HPV DNA type are summarized in Table 1. Passage 0 is the point at which cells were initially placed in culture, and one passage number was recorded each time a 70–80 % confluent dish of cells was passed. The passage number at which each isolate was analyzed in various experiments is indicated where relevant in the figures. All primary cervical carcinoma isolates were confirmed to express dramatically elevated levels of p16ink4a mRNA, a marker of cervical cancer (Sano et al., 1998), when compared to primary keratinocytes and fibroblasts (data not shown).
Exogenous expression of the BPV E2 gene (designated E2) in several different established cervical carcinoma cell lines causes irreversible growth arrest and senescence due to transcriptional repression of the HPV E6 and E7 genes (Goodwin et al., 2000; Wells et al., 2000). Previously we used an SV40-based recombinant virus vector (designated Pava) to express the wild-type (WT) E2 gene in HeLa-Sen2 cells, a strain of HeLa cells that is efficiently infected with this virus (Goodwin et al., 2000; Hwang et al., 1993). In this vector, the SV40 large T antigen gene is replaced with the E2 gene, but the SV40 capsid protein genes are retained. Replication-defective Pava virus particles containing the E2 gene packaged in an SV40 capsid are generated in permissive monkey CMT4 cells, which supply SV40 replication functions in trans (Settleman and DiMaio, 1988). Infection of HeLa-Sen2 cells with Pava delivers the E2 gene into essentially every cell, which leads to profound repression of E6/E7 and a rapid induction of senescence (Goodwin et al., 2000). Strikingly, although Pava caused dramatic growth arrest in HeLa-Sen2 cells, it had little or no effect on DNA synthesis of CaSki and SiHa cells, two cervical carcinoma cell lines that contain HPV16 DNA (Fig. 1A, grey bars). Pava infection also failed to inhibit DNA synthesis of all primary cervical carcinoma isolates tested, except CVX-102 (data not shown).
Because the introduction of the E2 gene into CaSki and SiHa cells by transfection or adenovirus vectors leads to growth arrest (Lee et al., 2002; Wells et al., 2000), we hypothesized that the SV40-based vector used to deliver the E2 gene did not efficiently infect these cells. In order to test this hypothesis, we measured the ability of WT SV40 to infect the three established cervical cancer cell lines. HeLa-Sen2, CaSki, and SiHa cells were infected with SV40 at an MOI of 10 (titer determined in permissive monkey cells), and infection was measured by immunostaining and flow cytometry of large T antigen expression. Typical histograms of flow cytometry data are presented in supplementary Figure S1. As predicted, more than 90 percent of HeLa-Sen2 cells expressed large T antigen (Fig. 1B, grey bars). However, less than 25 percent of CaSki or SiHa cells expressed large T antigen at this MOI. The inability of SV40 to efficiently infect SiHa and CaSki cells suggests that these cells also infect poorly with Pava, providing an explanation to their resistance to Pava-induced growth arrest.
We hypothesized that inefficient infection of CaSki and SiHa cells was a consequence of low levels of GM1, a cellular receptor used by SV40 (Tsai et al., 2003), on the surface of these cells. To test the levels of GM1 at the surface of cervical carcinoma cell lines, GM1 surface expression was measured with fluorescently-labeled cholera toxin B (CTXB), a bacterial toxin subunit which binds to GM1 (Chinnapen et al., 2007). Binding to intact cells was conducted at 4°C to prevent internalization and was measured by flow cytometry. SiHa and CaSki cells showed markedly lower levels of CTXB binding than HeLa-Sen2 cells (Fig. 1C, top), consistent with our suggestion that low levels of surface GM1 may limit SV40 infection in SiHa and CaSki cells. Addition of GM1 to the culture medium of murine cells that lack endogenous GM1 results in the incorporation of GM1 into the plasma membrane and a dramatic increase in the number of cells susceptible to SV40 infection (Schwarzmann, 2001; Tsai et al., 2003). As shown in Fig. 1C, bottom, addition of GM1 increased CTXB binding in Hela-Sen2, CaSki, and SiHa cells so that the level of CTXB binding was high in all cell lines.
We then tested whether elevating GM1 surface levels increased the ability of SV40 to infect CaSki and SiHa cells. Cells were treated with GM1 or left untreated. After washing away unincorporated GM1, cells were infected with SV40 at an MOI of 10, and infection was measured by assessing large T antigen expression by flow cytometry. As shown in Figure 1B (white bars), increasing the level of cell surface GM1 dramatically increased the ability of SV40 to infect CaSki and SiHa cells. In order to determine whether incorporation of GM1 also increased Pava infection, we measured E2 mRNA levels in untreated or GM1-treated cells. Pava-infected CaSki and SiHa cells displayed increased E2 mRNA expression after treatment with GM1, indicating that GM1 addition to cells increased Pava infection (Fig. S2.)
We also measured the effect of increased Pava infection on cell physiology. E2 expression in GM1-treated cells led to a 6- to 20-fold repression of HPV16 E6/E7 mRNA in CaSki and SiHa cells when compared to infected, untreated cells (Fig. 1D). We next tested whether Pava infection inhibited the DNA synthesis of GM1-treated cells. In contrast to the minimal effects of Pava infection in native CaSki and SiHa cells, infection of these cells with Pava following GM1 treatment led to a substantial repression of DNA synthesis to less than 3% of the mock-infected cells (Fig. 1A, white bars). Pava infection of GM1-treated CaSki and SiHa cells also led to a loss of the inactive hyperphosphorylated form of p105Rb and an increase in the active hypophosphorylated form (Fig. S3). Pava infection also increased total levels of p53 protein in CaSki cells (Fig. S3). The effects are more pronounced after GM1 addition. Although E2 expression did not appear to increase p53 levels in SiHa cells, an increase in p53 may occur prior to the 48hr time point at which protein was harvested (Goodwin and DiMaio, 2000). Taken together, these results indicated that the range of cells susceptible to efficient infection by SV40 and SV40 vectors can be expanded by addition of GM1. Therefore, we tested whether we could use this approach to express E2 in primary cervical carcinoma cells to determine the effect of E6 and E7 repression on cell growth.
We first used CTXB binding to measure GM1 surface expression in four HPV-positive primary human cervical carcinoma cell isolates and two primary cell isolates that lack HPV DNA, human foreskin fibroblasts (HFF) and keratinocytes (HFK). As shown in the top panels of Figure 2A, these cells displayed a wide range of GM1 surface expression. Addition of GM1 to the culture medium caused a marked increase in CTXB binding in all cells tested except CVX-102 cells, which displayed high basal CTXB binding (Fig. 2A). SV40 infection was measured as described above in untreated cells or cells treated with GM1. The high levels of endogenous GM1 in CVX-102 cells allowed efficient SV40 infection even without GM1 addition. In all other cell isolates, addition of GM1 to cells caused a dramatic increase in the fraction of cells expressing large T antigen after infection, approaching 90% in most cases (Fig. 2B).
Improved SV40 infection allowed us to investigate the effect of E2 expression on the proliferation of primary cervical carcinoma cells. HPV negative HFFs and HFKs were infected in parallel as controls. Cells were first treated with GM1 overnight to maximize Pava infection and delivery of the E2 gene. After removing unincorporated GM1, cells were infected with Pava, and DNA synthesis was measured 48 hrs post-infection. Expression of E2 caused a 6–20 fold reduction in DNA synthesis in the primary carcinoma cell lines tested, but had a minimal effect on DNA synthesis in HPV-negative cells (Fig. 3A). qRT-PCR of E2 mRNA levels demonstrated that differences in E2 expression did not dictate differences in DNA synthesis among the cell lines, because HFKs expressed at least as much E2 mRNA as any of the primary carcinoma cells (Fig. S4). These results indicated that growth arrest induced by E2 expression is specific to HPV-positive cells.
To begin to determine whether the observed repression of DNA synthesis was the result of E6 and E7 repression in primary cervical carcinoma cells, CVX-101, CVX-104, HFF, and HFK were treated with GM1 and infected with Pava expressing the WT E2 protein or an E2 mutant, K339M, defective for DNA binding. Reduced DNA binding by K339M E2 inhibits transcriptional repression of E6 and E7 (Goodwin et al., 1998; Prakash et al., 1992). The K339M E2 protein had little effect on DNA synthesis in CVX-101 and 104 cells when compared to the WT E2 protein, suggesting that the DNA binding activity of E2 is required for its ability to induce growth arrest (Fig. 3B). Similar results were also obtained with CVX-102 cells (data not shown). Neither WT nor K339M E2 caused significant inhibition of DNA synthesis in HPV-negative cells. The minor inhibition observed in HFKs suggested that Pava infection and/or E2 expression caused a minor, non-specific inhibition of DNA synthesis in HPV-negative cells, independent of the DNA binding activity of the E2 protein (Desaintes et al., 1999) (Fig. 3B). qRT-PCR for E2 mRNA expression confirmed that differences in growth inhibition between cells infected with WT or K339M Pava were not the result of differences in E2 mRNA levels, because K339M E2 mRNA levels were approximately equal to or greater than that of WT E2 (Fig. S5). Taken together, these data imply that the growth repression caused by E2 expression in primary cervical carcinoma cells is dependent upon the transcriptional repressor activity of the E2 protein and is likely the result of E6 and E7 repression.
In order to test whether earlier passage cells were also dependent on E6 and E7 for growth, we measured the effect of the E2 protein on CVX-106 cells at passage 3. Cells were treated with GM1 and infected with Pava expressing the WT or K339M E2 protein as described above. Due to the limiting number and slow growth of these early passage cells, DNA synthesis was measured at 72 hrs post infection in order to maximize the effect of E2 expression on growth. Even at these very early passages, WT E2 repressed DNA synthesis to a substantially greater degree than K339M E2 (Fig. 3C). These data indicated that extremely early passage primary cervical carcinoma cells are also dependent on E6 and E7 for continued growth.
To determine whether the WT E2 protein repressed E6 and E7 mRNA expression in primary carcinoma cells, qRT-PCR was used to measure endogenous HPV mRNA levels in two representative cervical carcinoma cell strains, CVX-104 and CVX-106. WT E2 repressed endogenous levels of HPV18 E6/E7 17-fold in CVX-104 cells and HPV16 E6/E7 levels 15-fold in CVX-106 cells, where as K339M E2 failed to repress 18E6/E7 in CVX-104 cells and repressed 16E6/E7 only three-fold in CVX-106 cells (Fig. 4A). In order to determine whether repression of E6 and E7 by E2 led to the reactivation of the p105Rb and p53 tumor suppressor pathways in primary carcinoma cells, protein was isolated from infected and mock-infected CVX-104 and CVX-106 cells and analyzed by immunoblotting. WT E2 caused an increase in p53 levels and the hypophosphorylation/activation of p105Rb in CVX-104 and CVX-106 cells, but K339M had no effect on p53 levels and/or the phosphorylation state of p105Rb (Fig. 4B). These results indicated that repression of E6 and E7 by the E2 protein led to the reactivation of p53 and p105Rb pathways in primary cervical carcinoma cells.
Repression of HPV E6 and E7 in HeLa and other established cervical carcinoma cell lines induces senescence, a form of irreversible growth arrest (Campisi and d'Adda di Fagagna, 2007; Goodwin et al., 2000; Wells et al., 2000). One biomarker used to distinguish senescence from other forms of growth arrest is an increase in senescence-associated (SA)-β-galactosidase activity (Dimri et al., 1995). In order to determine whether repression of E6 and E7 induced senescence in primary cervical carcinoma cells, CVX-104, CVX-106, and HFK cells were treated with GM1 and infected with Pava. To decrease the number of cells that escaped infection, cells were treated with GM1 and infected with Pava a second time. Nine days after the initial Pava infection, SA-β-galactosidase activity was assessed by staining with a colorimetric substrate. WT E2 expression increased SA-β-galactocidase staining in CVX-104 and 106 cells but not in HPV-negative HFK cells (Fig. 5A). Cells that expressed SA-β-galactocidase also displayed an enlarged and flattened morphology, which is characteristic of senescent cells (Fig. 5A). As expected, K339M E2 did not induce SA-β-galactosidase activity or these morphological changes, suggesting that these effects required repression of E6 and E7.
In addition to increased SA-β-galactosidase activity, senescence is also characterized by an increase in autofluorescence due to the accumulation of the fluorescent pigment lipofuscin (von Zglinicki et al., 1995). Following two rounds of GM1 treatment and infection with Pava, autofluorescence was measured by flow cytometry seven days after the initial infection with Pava (Fig. 5B). WT E2 and K339M E2 expression had no effect on autofluorescence in HFK cells, which continued to proliferate. Expression of the WT E2 protein, but not K339M E2, induced an increase in autofluorescence in a large fraction of CVX-104 and CVX-106 cells. Although a subpopulation of CVX-104 cells displayed low autofluorescence, we believe that these are cells that escaped infection with Pava and continued to proliferate during the seven day course of the experiment. This proliferating population is not seen in CVX-106 cells. However, since CVX-106 cells grow significantly slower than CVX-104 cells (data not shown), cells that escaped infection would still be a very minor fraction at day seven post-infection. Taken together, these data demonstrated that repression of E6 and E7 induces senescence in primary cervical carcinoma cells.
We performed a gene rescue experiment to confirm that the phenotypes elicited by Pava infection in primary cervical carcinoma cells were due to E6/E7 repression. CVX-104 cells were infected either with an empty retroviral vector or the vector encoding HPV16 E6 and HPV18 E7. Since these genes are expressed from the retroviral LTR, E2 does not affect their expression. After confirming expression of these genes by qRT-PCR (data not shown), the cells were treated with GM1 and infected with Pava. Growth arrest was then measured by DNA synthesis. As shown in Figure 6A, expression of exogenous HPV16 E6 and 18 E7 largely abrogated the inhibition of DNA synthesis caused by E2 expression in these cells. qRT-PCR confirmed that Pava-infected 16E6/18E7 expressing cells contained at least the same amount of E2 mRNA as infected control cells (data not shown). qRT-PCR using primers specific for the endogenous or exogenous E6/E7 transcripts confirmed that expression of the endogenous E6 and E7 genes in 16E6/18E7 cells was repressed by Pava infection to at least the same level seen in control cells (Fig. 6B), but expression of the exogenous E6/E7 genes was not affected (data not shown). Similarly, as measured by autofluorescence and granularity, which are both increased during senescence, there was a four- to five-fold reduction in the number of CXV-104 16E6/18E7 cells that senesced following Pava infection compared to infected control CVX-104 cells (Fig. S6). These data showed that E2-induced growth arrest and senescence in CVX-104 cells was the result of E6 and E7 repression.
The experiments reported here demonstrate that HPV oncogene expression is required for the proliferation of primary human cervical cancer cells in culture and provide insight into factors that control tropism of SV40 and SV40-based vectors. Numerous laboratories have demonstrated that established human cervical carcinoma cells lines depend on HPV E6 and E7 for growth or survival (Francis, Schmid, and Howley, 2000; Goodwin and DiMaio, 2000; Hwang et al., 1993; Jiang and Milner, 2002; Parish et al., 2006), but these previous experiments did not determine whether this requirement was an intrinsic property of cervical cancer cells or whether it was acquired during the establishment or propagation of permanent cell lines. Because established cancer cell lines, especially HeLa cells, diverge widely from tumors and primary cancer cells (Carlson, Iyer, and Marcotte, 2007; Sandberg and Ernberg, 2005), it seemed prudent to examine the requirement of E6/E7 in low passage human cancer cells. Furthermore, E6/E7 expression induces human keratinocytes to become resistant to terminal differentiation caused by high calcium and FBS (Munger et al., 1989; Pei et al., 1998; Schlegel et al., 1988; Sherman and Schlegel, 1996), the conditions typically used to culture established cell lines. Therefore, these conditions may allow the proliferation of only those cell that express E6 and E7. If this were the case, E6/E7 dependence is not an intrinsic property of cervical cancer cells, but rather an artifact of the cell culture conditions.
To determine whether cervical cancer cells have an intrinsic requirement for HPV E6 and E7, we examined the behavior of low passage cervical cancer cells cultured under conditions that do not select for E6 and E7 expression. We isolated cells from primary cervical cancers, cultured them in serum-free keratinocyte media with low calcium, and, after a minimal number of passages, analyzed their dependence on HPV E6 and E7. We believe that the early passage numbers at which these cells were tested and the conditions used to maintain them provide a better model for studying E6 and E7 dependence than established cell lines or cells immortalized in vitro. Unfortunately, the original tumors from which our primary cervical cancer strains were isolated no longer exist, so we cannot directly compare them to the primary cell strains analyzed here.
Expression of BPV E2 in all four primary cervical carcinoma cell strains repressed E6 and E7 and caused a significant inhibition of DNA synthesis (6- to 20-fold). Cancer cells as early as three passages after isolation displayed this response. E2 expression had only a slight effect on DNA synthesis of HPV-negative, primary HFFs and HFKs, and a DNA binding-defective E2 mutant had a minimal effect on DNA synthesis in the cancer cells. Moreover, growth arrest was abrogated by exogenous copies of E6 and E7. These results demonstrate that the ability of the E2 protein to bind to and repress the E6/E7 promoter is essential for its ability to induce growth arrest in primary human cervical cancer cells. Taken together, our results indicate that these primary human cervical cancer cells are dependent on E6 and E7 as early as three passages after isolation from patients and suggest that some in vivo human tumors are also dependent on E6/E7 for growth. However, even though the earliest passage cells we tested were highly dependent on HPV E6/E7, we have not ruled out the possibility that time in culture affects the sensitivity of cervical cancer cells to E6/E7 repression.
Repression of the E6 and E7 genes by the E2 protein in CVX-104 and CVX-106 cells led to elevated levels of p53 and active, hypophosphorylated p105Rb. Levels of total p105Rb were not elevated in the primary cervical cancer cells, unlike the situation in the established cell lines, highlighting a biochemical difference between primary cells and established cancer cell lines. In addition, E6/E7 repression in the primary cells resulted in growth arrest, increased autofluorescence and SA-β-galactosidase activity, and cell enlargement and flattening, all markers of cellular senescence. These data imply that repression of E6 and E7 in these primary cells induces senescence as a result of reactivation of p53 and p105Rb, as is the case in HeLa cells (DeFilippis et al., 2003). Senescence in this system is not due to up-regulation of p16ink4a, which is constitutively up-regulated in proliferating cervical cancer cells (Sano et al., 1998), including the primary cells studied here (unpublished data). Since senescence is irreversible (Campisi and d'Adda di Fagagna, 2007), we infer that repression of E6 and E7 in primary cervical carcinoma cells induces permanent growth arrest.
Lambert and colleagues demonstrated that repression of HPV16 E7 in a transgenic mouse model of cervical cancer caused tumor regression (Jabbar et al., 2009). Our results extend these findings from mice to human cells from invasive cancers that had undergone the decades-long process of carcinogenesis in women. The cellular basis of tumor regression in the mouse system has not been established, but in human cervical cancer cells, HPV repression causes senescence, which, like cell transformation, is under markedly different control in human and mouse cells (Balmain and Harris, 2000; Chaturvedi et al., 2004; Dotto, 1998; Goodwin et al., 2000; Newbold, 1997; Rangarajan et al., 2004). Nevertheless, the dependence on HPV oncogenes in primary human cervical cancer cells, established cervical cancer cell lines (and HPV-associated head-and-neck cancer cell lines (Rampias et al., 2009)) and transgenic mice indicates that oncogene dependence is a fundamental feature of HPV-induced cancers.
It was previously shown that human keratinocytes immortalized in vitro with transfected HPV16 DNA and maintained in the absence of FBS and in low concentrations of calcium are dependent on E6 and E7 for continued growth (Lee et al., 2002). However, immortalized cells are very different from cervical carcinoma cells. For example, unlike cells derived from tumors, cells immortalized in vitro by HPV are not tumorigenic in experimental animals (Durst et al., 1987; Lee et al., 2002; Pirisi et al., 1987; Woodworth et al., 1988). Furthermore, the vast majority of cervical high-risk HPV infections do not progress to cancer, and when progression does occur, it typically takes many years. These findings indicate that E6/E7 expression is not sufficient for tumorigenesis and that additional factors and genetic alterations are required for tumor formation in women. Thus, while it is not surprising that cells recently immortalized with E6/E7 in vitro retain their dependence on the proximal immortalizing agent, the relevance of this finding to human cancer cells is unclear.
It is striking that cancer cells retain E6/E7 dependence despite the onslaught of mutagenesis engendered by the viral oncoproteins. Remarkably, this dependence on E6/E7 extends to established cervical cancer cell lines such as HeLa, SiHa, CaSki, which have been exposed to decades of stress and mutagenesis during time spent in culture. It is possible that the viral oncogenes are more effective than acquired mutations at inactivating cellular tumor suppressor pathways or activating mitogenic pathways, thereby eliminating selective pressure for mutations in these pathways. However, expression of dominant-negative p53 blocks p53 signaling more completely than expression of HPV E6 (Butz et al., 1995). Rather, we propose that sustained dependence on the viral oncogenes is a consequence of the multiplicity of pathways that are targeted by the viral oncoproteins. For example, in addition to the p53 pathway, the E6 protein targets telomerase, several PDZ domain-containing proteins, and components of the DNA repair and apoptosis machineries (Howie, Katzenellenbogen, and Galloway, 2009). Because these multiple pathways are unlikely to be activated or inactivated by mutations in one or a few cellular genes, viral proteins are more effective drivers of cell growth and survival than are random mutations occurring in cellular DNA. This implies that therapies directed against individual cellular pathways targeted by the viral oncoproteins are likely to be less effective than therapies against the viral oncogenes themselves.
We have been able to expand primary cultures with high purity from 20–30% of cervical cancer tumor biopsies. Although this rate of success is higher than in a previously reported attempt to culture cervical carcinoma cells (Ku et al., 1997), we do not know if cervical carcinomas that fail to grow in culture also depend on E6 and E7. Nevertheless, our data indicate that E6 and E7 dependence is an intrinsic property of cervical carcinoma cells from at least a substantial fraction of patients and is not a trait that is acquired during the establishment and propagation of permanent cell lines. These findings in turn imply that treatments that inhibit the expression or activity of E6 and E7 may have therapeutic benefit in cervical cancer patients. The cancer isolates we studied were from early stage carcinomas; additional studies should determine whether cells isolated from advanced cervical carcinomas are also dependent on E6 and E7 for growth.
Our ability to infect these primary cancer cells with our E2 vector was informed by an understanding of the factors that control SV40 infection. The efficiency of SV40 infection appears to be dictated in large part by levels of cell-surface GM1, which binds directly to the major viral capsid protein, VP1 (Campanero-Rhodes et al., 2007; Neu et al., 2008; Tsai et al., 2003). In our experiments, CVX-102 and HeLa-Sen2 cells, which displayed the highest level of GM1, were efficiently infected with SV40 and Pava, whereas cells with lower levels of GM1, such as SiHa and CaSki cells and most primary cervical cancer cells, were infected poorly unless supplemented with GM1. These results demonstrated that infection by SV40 vectors can be improved by addition of GM1 to human cells.
There are reports that recombinant SV40 vectors and in vitro assembled SV40 virus-like particles are capable of efficiently transducing many mammalian cell types (Kimchi-Sarfaty et al., 2004; Lund et al., 2005; Strayer et al., 2005). However, by using quantitative measures of early viral gene expression, we found considerable differences in the ability of different cells to be infected by SV40 and SV40-based vectors generated in permissive monkey cells. In fact, seven of nine cell strains tested in this study infected poorly in the absence of GM1 supplementation. Similar differences have been documented by other groups in studies of the mechanism of SV40 infection, which identified GM1 as a cell surface receptor for this virus (Campanero-Rhodes et al., 2007; Low et al., 2004; Tsai et al., 2003). Our demonstration that different cell isolates display marked differences in GM1 cell-surface levels provides an explanation, at least in part, for these differences in infectivity. The addition of GM1 to the culture medium provides a simple and inexpensive method to expand the tropism of SV40 and SV40-based vectors for gene transfer and gene therapy applications.
CV-1, 293-T, and SiHa cells were purchased from American Type Culture Collection (ATTC, Manassas, VA). CV-1 cells were cultured in Minimal Essential Medium (MEM) Eagle, with 10 % FBS, standard antibiotics, 10 mM L-glutamine, and 10 mM HEPES pH 7.2 (standard supplements). HeLa-Sen2 cells were described previously (Goodwin et al., 2000). Normal diploid human foreskin fibroblasts (HFF) were obtained from the Yale Skin Diseases Research Center (YSDRC). CMT4 cells were provided by Y. Gluzman. CMT-4, HeLa-Sen2, HFF, 293-T, and SiHa cells were cultured in Dulbecco's MEM (DMEM) with standard supplements. CaSki cells were obtained from Michael Reiss (University of Medicine and Dentistry of New Jersey) and cultured in RPMI-1640 with standard supplements. Primary human foreskin keratinocytes (HFK) were obtained from YSDRC and cultured in Epilife medium (Invitrogen, Carlsbad, CA) with 60 µM calcium chloride, human keratinocyte growth supplement (Invitrogen, Carlsbad, CA), and standard antibiotics in the absence of serum. Hybridomas expressing PAb 108 mouse anti-large T antigen (purchased from ATTC) and PAb 597 mouse anti-VP1 (obtained from Edward Harlow, Harvard Medical School)) were cultured in DMEM plus 20% FBS and standard supplements.
CVX-101 to CVX-106 cells were isolated from primary tumors as described previously (Santin et al., 2005). Briefly, viable tumor tissue was harvested from solid tumor biopsies under sterile conditions at room temperature. Samples were mechanically minced, washed with RPMI-1640, and then incubated on a magnetic stirring apparatus in 0.14% collagenase type I (Sigma-Aldrich Corp., St. Louis, MO) and 0.01% DNAse (Sigma, 2000 kU/mg) in RPMI-1640 for 2 hrs at 37°C or overnight at 4°C. Single cell suspensions were generated by filtering enzymatically-dissociated tumor through 150 µM nylon mesh, washing in RPMI-1640 plus 10% human AB serum (Gemini Bioproducts, Calabasas, CA), and culturing in keratinocyte serum-free media (KSFM) (Invitrogen, Carlsbad,CA) supplemented with 35–50 µg/ml bovine pituitary extract, 5 ng/ml recombinant human EGF, 90 µM calcium chloride, and standard antibiotics (KSFM+). We succeeded in establishing primary cervical carcinoma cell strains in approximately 20–30% of attempts. Purity and homogeneity of fresh tumor cultures were tested by morphology, immunohistochemistry staining and/or flow cytometry with antibodies against cytokeratins and p16ink4a (CDKN2). Only primary cultures that had at least 95% viability and contained >99% epithelial cells expressing p16 were used in the experiments described below. HPV genotyping was conducted as described previously (Santin et al., 2005). Primary cell strains were shown to be free of HeLa cell contamination by using a PCR-based assay to detect a novel HeLa cell-specific retro-transposon (Rahbari et al., 2009). All cell isolates and cell lines were confirmed to have originated from different patients by amplifying and sequencing five genomic regions known to contain several single nucleotide polymorphisms (SNPs). The protocol and results for this analysis are available upon request. Cells were also found to be free of mycoplasma by using Mycoscope PCR Detection Kit (Genlantis, San Diego, CA) according to the manufacturer's instructions. Passage 0 of primary cells is the point at which cells were placed in culture. A passage number was recorded each time a 70–80% confluent 10 cm dish of cells was passed 1:4. Cells were thawed, expanded, and analyzed at the earliest passage available.
To generate cells that stably express exogenous HPV16 E6 and HPV18 E7, CVX-104 cells were first infected with the concentrated LXSN or LXSN 16E6 retrovirus stocks (see below) in KSFM+ and selected with 35 µg/ml G418 for nine days. Cells were then infected with concentrated RVY or RVY 18E7 retrovirus and selected in 20 µg/ml hygromycin for 12 days. Following selection, cells were maintained in KSFM+ with 17.5 µg/ml G418 and 10 µg/ml hygromycin.
Monosialoganglioside GM1 from bovine brain was purchased from Sigma-Aldrich Corp. Alexa Fluor 488 donkey anti-mouse IgG (H+L) and Alexa Fluor 488 Cholera Toxin B were purchased from Invitrogen. Mouse monoclonal anti-human p105Rb and mouse monoclonal anti-human p53 were purchased from BD Pharmingen (San Jose, CA). Goat polyclonal anti-humanβ-actin was purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA). RNeasy kits were purchased from Qiagen (Valencia, CA). iScript cDNA synthesis and iQ SYBR Green Supermix kits were purchased from BioRad (Hercules, CA). Tritiated thymidine was purchased from MP Biomedicals (Solon,OH).
WT SV40 strain 776 was generated from pBRSV DNA (obtained from ATTC) in permissive CV-1 cells. A final high titer stock was generated using the Pava harvesting protocol as described previously (Goodwin et al., 1998; Naeger et al., 1999). Titers of WT SV40 stocks were determined by infecting CV-1 cells and measuring the fraction of cells expressing large T antigen (see below) at 24 hrs post infection by immunostaining and flow cytometry. Prior to the generation of SV40 virus from pBRSV DNA, a phenylalanine mutation at position 325 of VP1 found in pBRSV DNA was converted to WT serine by standard site-directed mutagenesis.
BPV1/SV40 recombinant viruses, designated Pava, which encode WT or K339M BPV-E2 were generated and harvested as described previously (Goodwin et al., 1998; Naeger et al., 1999). The E2 repressor and E5 genes were disrupted in WT Pava vector but remained intact in K339M Pava {described in (Goodwin et al., 1998)}. Virus stocks were titered by infecting CMT-4 cells with Pava and measuring the fraction of cells that expressed VP1 by immunostaining and flow cytometry. The protocol used for measuring VP1 expression is identical to the protocol used for measuring large T antigen expression (see below), except cells were fixed in 10% formaldehyde and permeabilized in 10% formaldehyde with 0.2% triton X-100. PAb 597 mouse anti-VP1 was used to measure VP1 expression.
Retroviruses expressing HPV16 E6 and HPV18 E7 were described previously (DeFilippis et al., 2003; Halbert, Demers, and Galloway, 1991). Retrovirus stocks were generated in 293-T cells as described previously (Yates et al., 2008). Virus was harvested at 48 hrs post transfection and concentrated with Peg-It (System Biosciences, Mountain View, CA) according to the manufacturer's instructions. Briefly, one volume of Peg-It was added to four volumes of retrovirus at 4°C overnight. Precipitated retrovirus was pelleted by centrifugation, washed, and resuspended in KSFM+.
To measure DNA synthesis, an equal number of cells were plated in triplicate. Cells were treated with 10 (HeLa, SiHa, CasKi) or 15 µg/ml GM1 (all other cells) overnight in either the medium used to culture cells (keratinocytes and CVX-101 to CVX-106 cells) or DMEM with 1% FBS (all other cells). Cells were then washed with PBS (keratinocytes and CVX-101 to CVX-106 cells) or DMEM with 10% FBS (all other cells lines) and either mock-infected or infected with Pava. At the indicated time post-infection, cells were treated with media containing 1.5 uCi of [3H]thymidine/ml for five to six hours. After precipitating nucleic acids with 10% trichloroacetic acid, DNA synthesis was determined by measuring the incorporation of [3H]thymidine into acid-insoluble material using a liquid scintillation counter.
For immunoblotting, cells were harvested at the indicated times post-infection and lysed on ice in NP40 buffer (50 mM TRIS pH 8.0, 1 mM EDTA, 150 mM NaCl, 1% NP40, 0.1% SDS, standard protease inhibitors) for 12 minutes. Debris was cleared by centrifugation, and equal quantities of protein were electrophoresed on standard 7.5% or 12% polyacrylamide gels containing SDS. After electrophoresis, proteins were transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA) in transfer buffer (12.5 mM Tris, 0.1 M glycine, 20% methanol) and blocked in 5% milk/TBST buffer (5% nonfat dry milk, 25 mM Tris-HCl [pH 8.0], 125 mM NaCl, 0.1% Tween-20). The membranes were probed with the following specific antibodies: mouse monoclonal anti-human p105Rb, mouse monoclonal anti-human p53, or goat polyclonal anti-humanβ-actin.
Quantitative real-time PCR (qRT-PCR) and primer design were performed as described previously (Johung, Goodwin, and DiMaio, 2007). Briefly, total RNA was harvested using the RNeasy with DNAse treatment or RNeasy Plus kit at indicated times post-infection. 1 µg RNA was used as a template for cDNA synthesis using an iScript cDNA synthesis kit. qRT-PCR was performed by using iQ SYBR Green Supermix with 40 ng cDNA per 20 µl reaction and the BioRad MyiQ Single-color Real-time PCR detection system. GAPDH transcripts were detected using primers 5'-CAGCCTCAAGATCATCAGCA-3' and 5'-TGTGGTCATGAGTCCTTCCA-3'. BPV-E2 transcripts were detected using 5'-GACGAGGCAGCCAGATTTAG-3' and 5'GGGTCTCCTTCAGGTCCTTC-3'. HPV16 E6/E7 transcripts were detected using primers 5'-ACAAGCAGAACCGGACAGAG-3' and 5'-GCCCATTAACAGGTCTTCCA-3', which annealed to the E7 ORF present in both transcripts. HPV18 E6/E7 transcripts were detected using primers 5'-TGAAATTCCGGTTGACCTTC -3'and 5'-CACGGACACACAAAGGACAG-3', which annealed to the E7 ORF present in both transcripts. Exogenous HPV18 E7 transcripts were detected using primers 5'-AAGCTCAGCAGACGACCTTC-3' and 5'-CACAGCCGGATCAGCTTACT-3', which annealed to the E7 ORF and the 3'UTR of the RVY vector. Endogenous HPV18 E6/E7 transcripts were distinguished from exogenous HPV18 E7 transcripts by using primers 5'-CCAGAAACCGTTGAATCCAG-3'and 5'-GTTGGAGTCGTTCCTGTCGT-3', which annealed to the HPV18 E6 ORF present in both endogenous transcripts but absent from the exogenous E7 ORF.
Cells were treated with GM1 as described above and infected with SV40 at the indicated MOI. At 48 hrs post-infection, cells were trypsinized, neutralized, and washed once with PBS. Cells were then fixed by adding ice cold methanol dropwise to the cell pellet while vortexing gently, and the fixation continued for at least 20 minutes on ice. After centrifugation, methanol was removed, and cells were blocked for 5 min in PBS plus 0.5% bovine serum albumin (BSA). The cell pellet was then resuspended in 100 µl of a 1:1 mixture of 5% normal donkey serum in PBS (NDS-PBS) to PAb 108 monoclonal mouse anti-large T antigen supernatant and incubated for 1 hr at 37°C. The cells were then washed twice with PBS + 0.5% BSA and resuspended in 100 µl NDS-PBS containing a 1:500 dilution of Alexa Fluor 488 donkey anti-mouse IgG and incubated at 37°C for 30 minutes. Following two washes in PBS + 0.5% BSA, the cell pellet was resuspended in 200 µl PBS and kept on ice until analysis. The fraction of cells expressing large T antigen was measured using a 488 nm excitation and 530 nm emission filter on a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA) and plotted on a 650 nm vs 530 nm emission filter 2D-density plot.
Cells were mock-treated or treated with GM1 as described above. After removing unincorporated GM1, cells were trypsinized, neutralized, and washed once with ice cold PBS. Cells were then resuspended in 500 µl of ice cold DMEM with 1 µg/ml Alexa Fluor 488 labeled cholera toxin B (CTXB) and incubated at 4°C for 30 min. Cells were then washed twice in cold PBS, resuspended in PBS, and kept on ice until analysis. CTXB binding was analyzed using a 488 nm excitation and 530 nm emission filter on a FACS Calibur flow cytometer.
To assess autofluoresence and/or granularity, cells were treated with GM1 and infected with Pava at the indicated MOI as described above. Where indicated, the process was repeated a second time to reduce the number of cells that escaped infection. At the indicated times after the first infection with Pava, autofluoresence was measured by flow cytometry of unstained cells using 488 nm excitation and a 650 nm emission filter on a FACS Calibur flow cytometer. Granularity was measured using a 488 nm laser and side scatter channel (SSC). For measuring senescence-associated (SA)-β-galactosidase, cells were treated with GM1 and infected with Pava twice as above. Nine days after the first infection, SA-β-galactosidase staining was performed as described previously (Dimri et al., 1995; Goodwin et al., 2000).
A two-tailed T-test was used for all statistical analysis of data.
Low passage human cervical carcinoma cell lines maintained in serum-free medium in low calcium require sustained expression of the human papillomavirus oncogenes for continuous proliferation.
Extinction of HPV oncogene expression in these cells causes cellular senescence. Incubation of primary human cells with the ganglioside GM1 is a simple method to improve infection efficiency by SV40 and SV40-based viral vectors.
We thank Ivan Martinez and Edward Goodwin for advice and reagents, Daniel Dykas for analysis of SNPs, and Jan Zulkeski for assistance in preparing this manuscript. T.G.M. was supported in part by a training grant from the National Institutes of Health. L.L.A. was supported in part by a training grant and an individual National Research Service Award from the National Institutes of Health. This work was supported by a grant from the National Cancer Institute to DD (P01 CA016038).
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text:
Restoration of normal glucose tolerance in severely obese patients after bilio-pancreatic diversion: role of insulin sensitivity and beta cell function
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The aim of this study was to analyse the mechanisms underlying the improvement in glucose tolerance seen in morbidly obese patients undergoing bilio-pancreatic diversion (BPD).
Subjects and methods
We evaluated glucose tolerance (by OGTT), insulin sensitivity (euglycaemic–hyperinsulinaemic clamp and the OGTT index OGIS) and beta cell function (OGTT modelling analysis) in 32 morbidly obese (BMI=52±7 kg/m2, mean±SD) patients (12 with NGT, 9 with IGT and 11 with type 2 diabetes), before and after BPD, and in 22 lean control subjects. Patients were studied before and from 7 days to 60 months after surgery.
BPD improved glucose tolerance in all subjects, who after surgery all had normal glucose tolerance. Insulin sensitivity was restored to normal levels in all subjects (pre-BPD 341±79 ml min−1 m−2, post-BPD 511±57 ml min−1 m−2, lean 478±49 ml min−1 m−2). The insulin sensitivity change was detectable within 10 days of BPD. At baseline, beta cell sensitivity to glucose was impaired in diabetic subjects (25 [18] pmol min−1 m−2 l mmol−1, median [interquartile range]) compared with lean subjects (82 [98]; p≤0.05). After BPD, beta cell glucose sensitivity showed a tendency towards improvement but remained impaired in diabetic subjects (30 [62]; p<0.01 vs lean). Total insulin output decreased in parallel with the insulin sensitivity increase in all groups. In the whole patient group, mean OGTT glucose levels were inversely related to both insulin sensitivity and beta cell glucose sensitivity (r2=0.67, partial r=−0.76 and −0.41, respectively). NEFAs, leptin and adiponectin were related to insulin sensitivity but could not explain the early improvement.
Following BPD, glucose tolerance was restored mainly as a result of a rapid and large improvement in insulin sensitivity.
KeywordsBariatric surgery Beta cell function Bilio-pancreatic diversion Glucose tolerance Insulin resistance Insulin secretion Insulin sensitivity Morbid obesity Type 2 diabetes
bilio-pancreatic diversion
oral glucose insulin sensitivity
Recent experience with bariatric surgery for severe obesity has shown that this form of treatment can restore normal glucose tolerance in the majority of diabetic subjects [1]. Two surgical procedures, bilio-pancreatic diversion (BPD) and Roux-en-Y gastric bypass, have proven so effective in restoring normoglycaemia that they have been proposed as potential therapies for diabetes [2, 3].
In morbidly obese patients undergoing bariatric surgery, one factor contributing to the restoration of normal glucose tolerance is the surgery-induced increase in insulin sensitivity [4, 5, 6, 7]. Although some data are available [5, 7, 8, 9], the role of beta cell function, its relationship with insulin sensitivity, and the contribution of either factor to the improvement in glucose tolerance are not clearly established. Furthermore, while there is evidence that the improvement in circulating glucose levels occurs in days [10], it is not clear how early after surgery the improvement in insulin sensitivity occurs.
In this study, we used an OGTT and validated insulin sensitivity and beta cell function methods in severely obese subjects, covering a wide spectrum of glucose tolerance, who underwent BPD to elucidate the role of insulin sensitivity, beta cell function, and their interplay in the restoration of glucose tolerance.
Subjects and methods
Anthropometric and metabolic characteristics
Control subjects
Type 2 diabetes
BPDa effect
Sex (male/female)
Age (years)b
Weight (kg)
BMI (kg/m2)
Mean glucose (mmol/l)
Fasting insulin (pmol/l)
38 (32)
114 (68)c
42 (21)
108 (101)c
43 (29)
106 (56)c
42 (22)
This malabsorptive surgical procedure [11] consists of an approximately 60% distal gastric resection with stapled closure of the duodenal stump. The residual volume of the stomach is about 300 ml. The small bowel is transected at 2.5 m from the ileo-caecal valve, and its distal end is anastomosed to the remaining stomach. The proximal end of the ileum, comprising the remaining small bowel (involved in carrying bilio-pancreatic juice but excluded from food transit), is anastomosed in an end-to-side fashion to the bowel, 50 cm proximal to the ileo-caecal valve. Consequently, the total length of absorbing bowel is reduced to 250 cm, the final 50 cm of which, the so-called common channel, represents the site where ingested food and bilio-pancreatic juices mix.
Body composition
Body weight was measured to the nearest 0.1 kg with a beam scale, and height to the nearest 0.5 cm using a stadiometer (Holtain, Crosswell, UK). Lean body mass, in kg, was calculated from total body water, determined by tritiated water injection [12]. Fat mass was then calculated as the difference between body weight and lean body mass. When determined very early (up to 1 month) after surgery, body composition was measured by electrical bioimpedance (Dietosystem, Milan, Italy).
A standard 75-g OGTT was performed in each patient at baseline and after surgery, with blood sampling at 0, 30, 60, 90, 120 and 180 min. In some tests (seven of the 64 tests in BPD subjects and 13 of the 22 tests in lean subjects), sampling was stopped at 2 h.
Euglycaemic–hyperinsulinaemic clamp
Peripheral insulin sensitivity was evaluated by the euglycaemic–hyperinsulinaemic technique [13] in a subset of the BPD patients (26 pre-BPD and 18 post-BPD determinations). After inserting a cannula in a dorsal hand vein for sampling arterialised venous blood, and another in the antecubital fossa of the contralateral arm for infusions, the subjects rested in the supine position for at least 1 h. They were placed with one hand warmed in a heated air box set at 60°C to obtain arterialised blood samples. Insulin sensitivity, as the total insulin-mediated glucose uptake (M value, in micromole per minute per kilogram lean body mass), was determined during a primed constant infusion of insulin of 240 pmol min−1 m−2. The M value was calculated during the last 40-min period of the clamp.
Insulin sensitivity and beta cell function
Insulin sensitivity was calculated from the OGTT as the oral glucose insulin sensitivity index (OGIS) [14], using the 2-h OGIS equation. This method provides an insulin sensitivity index that is an estimate of the glucose clearance during a euglycaemic–hyperglycaemic clamp, expressed in millilitre per minute per square metre of body surface area. In the subset of subjects in whom the clamp was also performed (44 tests), there was a strong correlation between the M value and OGIS (r=0.92, p<0.0001). A good correlation was also observed between the changes in OGIS and the M value occurring after BPD (r=0.68, p<0.005, n=17). Because the M value was not available in all subjects, we used OGIS to quantify insulin sensitivity in all subjects. Values obtained using this method show good correlation with those obtained by the clamp.
Beta cell function was assessed using a model describing the relationship between insulin secretion and glucose concentration, which has previously been explained in detail [15, 16]. The model expresses insulin secretion as a sum of two components. The first component represents the dependence of insulin secretion on absolute glucose concentration at any time point, and is characterised by a dose–response function relating the two variables. The dose–response is the mean slope within the observed glucose range, denoted as beta cell glucose sensitivity. The dose–response is modulated by a potentiation factor, which accounts for several potentiating factors (prolonged exposure to hyperglycaemia, non-glucose substrates, gastrointestinal hormones, neurotransmitters). The potentiation factor is set to be a positive function of time and to average one during the experiment. It thus expresses a relative potentiation of the secretory response to glucose. In previous experiments [16, 17], we have found that insulin secretion at the end of an OGTT or meal is relatively higher than at the beginning for comparable glucose levels (i.e. when glucose returns to the basal level, insulin secretion remains higher). Potentiation thus increases during the test; this increase has been quantified as the ratio between the potentiation factor value at 2 h after the OGTT and that at time zero [16, 17]. The second insulin secretion component represents a dynamic dependence of insulin secretion on the rate of change of glucose concentration. This component is termed derivative component, and is determined by a single parameter, denoted as rate sensitivity. Rate sensitivity is related to early insulin release [16, 17].
The model parameters were estimated from glucose and C-peptide concentration by regularised least-squares, as previously described [15, 16]. Regularisation involves the choice of smoothing factors selected to obtain glucose and C-peptide model residuals with standard deviations close to the expected measurement error (∼1% for glucose and ∼4% for C-peptide).
Basal and total insulin secretion during the OGTT were calculated from the estimated model parameters. Total insulin secretion was calculated as the integral of insulin secretion during the first 2 h of the OGTT, both in the 2- and 3-h OGTT protocols. Insulin secretion was expressed in picomole per·minute per·square metre of body surface area.
Statistical analysis
Data are presented as means±SD, unless otherwise stated. Insulin parameters, which, in general, are not normally distributed, are presented as medians (interquartile range). ANOVA was used to compare group values, with post hoc group comparisons tested by the Bonferroni–Dunn test. Treatment-induced changes in the obese group were tested by two-way ANOVA for repeated measures. Simple and multiple regression was carried out by standard techniques, using logarithmic transformation of non-normally distributed variables. A p value of ≤0.05 was considered statistically significant.
Weight and glucose tolerance
At baseline, compared with lean control subjects, obese patients had higher mean plasma glucose concentrations during the OGTT, the differences being larger in IGT and type 2 diabetic subjects (Table 1). After surgery, body weight and BMI were much reduced, but remained higher than in lean control subjects, particularly in IGT and type 2 diabetic patients. In contrast, mean plasma glucose concentrations were no longer higher than those of lean control subjects in the NGT, IGT or type 2 diabetes groups.
The impact of surgery on weight loss was strongly time dependent. Thus, in the patients examined 3 months or longer after surgery, weight loss averaged 37% (range 12–53%). In contrast, the weight loss in patients examined within 10 days of BPD was 6% at most. In the patient group as a whole, the relative weight loss was strongly and directly related to the time elapsed since surgery (r=0.83, p<0.0001, both variables log transformed).
Insulin sensitivity
Insulin sensitivity and beta cell function parameters
Control subjects
Type 2 diabetes
BPDa effect
Insulin sensitivity (ml min−1 m−2)
Fasting insulin secretion (pmol min−1 m−2)
67 (43)
168 (123)b
65 (23)
135 (102)b
57 (59)
121 (75)b
76 (51)
Insulin output (nmol/m2)
37 (18)
53 (22)b
26 (19)
46 (12)b
28 (20)
36 (13)
20 (9)b
Glucose sensitivity (pmol min−1 m−2 l mmol−1)
82 (98)
78 (71)
78 (52)
55 (34)
95 (92)
25 (18)b,c
30 (62)b,c
Rate sensitivity (nmol m−2 l mmol−1)
0.6 (1.0)
0.0 (0.7)
0.0 (0.3)
0.4 (1.1)
1.0 (1.1)
0.4 (0.8)
0.3 (0.5)
Potentiation ratio (fold)
1.3 (0.7)
1.2 (0.5)
1.5 (0.6)
1.3 (0.4)
1.1 (0.2)
1.1 (0.4)
1.0 (1.0)
Non-BPD surgical patients
In the group of subjects undergoing surgery for reasons other than BPD, the weight loss was 1.7±1.9 kg (∼1%). Insulin sensitivity did not change (373±80 vs 368±80 ml min−1 m−2 post-surgery) and the two insulin sensitivity measurements were strongly correlated (r=0.74, p<0.0001). In addition, glucose tolerance category was largely unaltered after surgery: the diabetic subjects remained diabetic (except one, who had a diabetic fasting glucose concentration and was classed as IFG after surgery). On average, surgery slightly worsened glycaemic control: mean post-OGTT glucose levels were a little higher after surgery than pre-surgery (8.8±3.3 vs 8.2±3.1 mmol/l, p<0.001). In parallel with this ∼8% increase, mean post-OGTT C-peptide increased slightly (by ∼5%, from 924±24 to 971±24 pmol/l, p<0.002).
Beta cell function
Before surgery, the fasting insulin secretion rate was increased in NGT, IGT and type 2 diabetic patients compared with lean control subjects, whereas beta cell glucose sensitivity was decreased in type 2 diabetes (Table 2). Following surgery, the fasting insulin secretion rate and total insulin output decreased. There was a tendency towards an improvement in beta cell glucose sensitivity in IGT and type 2 diabetes, but the change did not reach statistical significance. Furthermore, beta cell glucose sensitivity remained significantly impaired in type 2 diabetic patients compared with lean control subjects. No systematic between-group differences were found in terms of rate sensitivity or potentiation, either before or after surgery.
NEFA and adipokines
NEFA and adipocyte hormones
Control subjectsa
Type 2 diabetes
BPDb effect
Fasting NEFA (mmol/l)
Leptin (μg/ml)
Adiponectin (μg/ml)
12±2c, d
One important result of this study is that in severely obese patients with IGT or type 2 diabetes, BPD restored NGT and insulin sensitivity. Glucose tolerance and insulin sensitivity were significantly heightened post-surgery, even in patients who were NGT at baseline. Previous studies [4, 5, 6, 7] have shown that bariatric surgery is extremely effective in improving glucose tolerance, insulin sensitivity and, to some extent, insulin secretion (as inferred from homeostasis model assessment estimates or the acute insulin response to i.v. glucose). These studies, however, provided a limited description of the simultaneous changes in beta cell function and insulin sensitivity associated with severe obesity and their response to surgical treatment, particularly in the presence of defects in glucose tolerance. Our results show that those obese subjects who are IGT or have type 2 diabetes prior to surgery are characterised by impairment of beta cell glucose sensitivity. This result confirms previous findings that impaired beta cell glucose sensitivity is the main determinant of IGT and type 2 diabetes, rate sensitivity and potentiation being relatively minor factors [17, 18, 19]. The impairment in beta cell glucose sensitivity was seen together with a variable degree of insulin resistance and the attendant upregulation of insulin secretion (Fig. 3), as described previously [19]. After surgery, insulin sensitivity and insulin secretion showed reciprocal changes, as shown in Fig. 3.
Our data on beta cell function are not directly comparable with those obtained in previous studies [5, 8, 9], because of the different surgical techniques (variants of vertical banded gastroplasty) and beta cell function tests (arginine and i.v. glucose infusions). Those studies found a decrease in plasma insulin response and an increase in insulin sensitivity; a disposition index, calculated as the product of the two parameters, was either significantly increased after surgery [5] or unaltered [8, 9], suggesting improved or maintained beta cell function. However, the correct assessment of beta cell function changes, through parameters such as the disposition index, requires that beta cell function parameters be independent from glucose levels and clearly related to insulin sensitivity in a control group [20]. In our cohort of control subjects, the parameter that correlated with insulin sensitivity was total insulin output, which is, however, an index that closely depends on glucose levels. Thus, although our post-surgery findings show augmented insulin sensitivity and a reduced insulin secretory response to oral glucose (Fig. 3), precise assessment of the degree of compensation in the groups was not possible.
For similar reasons, the present results on beta cell function cannot be directly compared with those obtained in similar patients using an i.v. glucose test [7]. This study showed that bariatric surgery restored first-phase insulin secretion. In our beta cell model, the parameter that is related to acute insulin response, namely, rate sensitivity, was not significantly affected by treatment (Table 2). This could be due to the insufficient power of the OGTT to detect changes in acute insulin secretion [21], as also indicated by the large variability of the rate sensitivity parameter (Table 2) and its modest reproducibility [16], or to the intrinsic differences between the i.v. glucose test and the OGTT [19].
As expected from previous work [17, 18, 19], we found a strong inverse relationship between beta cell glucose sensitivity and glucose tolerance (as the 2-h plasma glucose concentration). This relationship was maintained after surgery, but became significantly steeper (Fig. 4), i.e. the same beta cell glucose sensitivity was associated with lower glucose levels after than before surgery. This probably reflects the contribution of improved insulin resistance to glucose tolerance. In fact, beta cell glucose sensitivity and insulin sensitivity together explained almost 70% of the variability in mean plasma glucose concentrations. Using the regression equation, a subject with the insulin sensitivity of the NGT group but the beta cell glucose sensitivity of the type 2 diabetes group would have a mean glucose level of 7.9 mmol/l, a value in the range of that for IGT subjects (7.7–10.3 mmol/l). Whether the pattern of glucose absorption, which appears to be complete [22], is a further determinant of glucose levels cannot be established from the results of the present study.
Another novel finding of the present study was that substantial improvements in mean post-OGTT plasma glucose concentrations (averaging 3.5 mmol/l) and insulin sensitivity occurred within days of surgery, before any significant weight loss. This early improvement could not be explained by changes in fasting plasma NEFA, leptin or adiponectin concentrations, which were minimal in the first 10 days. This early effect does not appear to be related to non-specific factors associated with surgery, such as acute reduction of nutrient intake. In fact, in the group of patients who underwent abdominal surgery for reasons other than BPD and received the same post-surgery nutrition regimen, insulin sensitivity–evaluated using the same technique as employed in the present study–was not different 4–10 days after surgery compared with before surgery, nor was glucose tolerance improved by surgery. The obvious limitation of our experimental design is that patients were not studied sequentially at multiple times following surgery. Nevertheless, a marked improvement in insulin sensitivity was observed in all eight of the patients studied within 10 days of surgery (one with NGT, four with IGT and three with type 2 diabetes).
It can be hypothesised that some unknown regulator of insulin sensitivity is directly affected by BPD. With regard to this, it should be noted that while several surgical techniques leave the duodenum in place, duodenal transit is excluded in the standard BPD. Indeed, exclusion of the duodenum from alimentary transit has been shown to be crucial for the reversion of diabetes in Goto–Kakyzaky rats [23]. Thus, some hitherto unknown substance with insulin desensitising properties would be produced in the duodenal tract of severely obese subjects upon nutrient passage; shunting food downstream to the duodenum would result in a fall in the release of such substance, with a consequent brisk improvement in insulin action.
We did not measure the classical gastrointestinal hormones potentially involved in glucose homeostasis (glucose-dependent insulinotropic peptide, glucagon-like peptide 1, ghrelin, and others), which are known to change after bariatric surgery and have been postulated to play a role in the insulin sensitivity increase (e.g. [24]). However, these hormones do not seem likely candidates, as there is no evidence that they can cause drastic changes in insulin sensitivity such as observed in this study. An effect on beta cell function is, on the other hand, possible.
Several relevant questions remain to be answered. Firstly, it would be important to test whether the severe beta cell glucose insensitivity of severely obese patients with type 2 diabetes eventually recovers up to normal levels. Secondly, it is not clear to what extent the severe obesity model reflects pathophysiological processes occurring in moderately obese or in non-obese subjects with IGT or type 2 diabetes. Finally, the nature and dynamics of putative factors released by the upper gastrointestinal tract must be investigated by appropriate experimental models in animals and clinical designs in humans.

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International comedy sensation and ventriloquist Jeff Dunham, along with his cast of hilarious characters, will return to Las Vegas by popular demand for 15 nights of their hugely successful "Perfectly Unbalanced" show.
Dunham's dedication to his craft and relentless innovation will be recognized as his one of a kind comedy empire has been nominated to the Hollywood Walk of Fame. Over the past 46 years, Dunham and his world famous characters; Walter, Achmed, Bubba J, Peanut and José Jalapeno have garnered numerous accolades; from being named Forbes' Celebrity 100 list of Most Powerful Entertainers, as well as setting the Guinness World Records for the "Most Tickets Sold for a Stand-Up Comedy Tour," to setting the record for viewership on Comedy Central.
Dunham has carved a unique corner in the comedy world that's as funny as it is popular. The New York Times Magazine declared Jeff Dunham "the most successful comedian working in America." He has been named Pollstar's #1 Comedy Tour in North America for three years running as well as their Top Worldwide Tour for two years in a row.
Orders of 10 or more can contact our Group Sales department at (866) 574-3851 or send us an email.


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Tommy Hilfiger Boys Printed Shirt, Navy

Create a smart causal look for your little man with this gorgeous printed skirt from Tommy Hilfiger in navy. Styled with a buttoned down collar and Hilfiger flag embroidered on the chest. It is crafted from a lightweight crisp cotton fabric which will look great with chinos and canvas trainers and is crafted in as custom fit for that tailored look.

Brand: Tommy Hilfiger

Style Code: THB02331

Colour: 431

Material: 100% Cotton

Wash Care: Machine Wash at 40°C

p-THB02331-431Tommy Hilfiger Boys Printed Shirt, Navy
€ 0.00 to 0.00


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Dating in Sun City for Sugar Babies and Sugar Daddies
Sun City, California is filled with singles, but how do you know they're going to offer you what you want and need? Are you a Sugar Baby looking for the generous and wealthy Sugar Daddy of her dreams, or are you a Sugar Daddy who needs a sexy and loyal Sugar Baby? Find the mutually beneficial relationship in Sun City that you deserve on SugarDaddyForMe today!


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There was a time when pairing two of golf's brightest lights was a rarity. Now it is becoming a weekly tee time.
And who can blame anyone for wanting Rory McIlroy and Tiger Woods to be in the same group.
That is enough wattage for television executives to break out into a dance.
TIGER WOODS: Talks about NFL toughness
GOLF: This week at a glance
McIlroy and Woods will join Nick Watney in the first two rounds of the BMW Championship. The threesome tops the FedExCup standings and tees off Thursday at 11:48 a.m. ET on No. 10 at Crooked Stick.
Other tee times to watch (courtesy of the Golf Channel):
11:48: Matt Kuchar, Hunter Mahan, Sergio Garcia (start on No. 1)
12:43: Bubba Watson, Carl Pettersson, Keegan Bradley (on No. 1)
12:54: Brandt Snedeker, Louis Oosthuizen, Dustin Johnson (on No. 1)
11:37: Jason Dufner, Zach Johnson, Phil Mickelson (on No. 10)
12:43: Justin Rose, Lee Westwood, Rickie Fowler (on No. 10)
12:54: Steve Stricker, Luke Donald, Ernie Els (on No. 10)
Reid Cherner has been with USA TODAY since 1982 and written Game On! since March 2008.
He has covered everything from high schools to horse racing to the college and the pros. The only thing he likes more than his own voice is the sound of readers telling him when he's right and wrong.
Michael Hiestand has covered sports media and marketing for USA TODAY, tackling the sports biz ranging from what's behind mega-events such as the Olympics and Super Bowl to the sometimes-hidden numbers behind the sports world's bottom line.


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War Fiction Novels
Margaret Pemberton's The Londoners trilogy is a saga set in the nation's capital that touches on many of the social issues that engulfed the country during the 1940s and 50s.
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The first book introduces Carl Voigt, a First World War prisoner of war who had married a Cockney girl and never moved back to his native Germany. Now a widower, Carl and Kate were always popular but during the Second World War, they start to be treated as outcasts. Finding friends is hard and Kate finds herself alone and pregnant. However, during the blitz Kate forms a bond with a wounded sailor called Leon Emmerson who is also being shunned by society...
The following books find Kate facing a battle just after the war to keep her young son with her; and her new family celebrating the Coronation in 1953.
Three novels full of adventure and intrigue in wartime Europe, these books by Alan Furst will appeal to fans of John Le Carre, Henry Porter and John Lawton.
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Kingdom of Shadows follows former Hungarian cavalry officer Nicholas Morath as he returns home to his mistress in 1938. While away in Vienna, he has done a quiet favour for his diplomat uncle. Desperate for Hungary to stop drifting into alliance with Nazi Germany, his uncle has been trading in conspiracies - meaning Morath is edging closer to danger...
Mission to Paris and The Foreign Correspondent are equally poignant and compelling, providing a remarkable insight into the frailties and fears of humanity when under pressure.
Chronicling some of the most astonishing chapters of our times, our three-book Max Hastings Collection brings together three very different - but equally compelling - analyses of modern history's most gripping military stories.
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The Battle for the Falklands takes a thoughtful look at one of the British military's most strange and unbelievable conflicts from a social, governmental and tactical standpoint; Bomber Command details the Allies' six-year air offensive against Germany; and the march of the 2nd SS Panzer Division through France to reinforce the front line defenders of Hitler's Fortress Europe is skilfully accounted in Das Reich.
Written by award-winning journalist and former Daily Telegraph and Evening Standard editor Max Hastings, this three-book military history collection will make gripping reading for the armchair historian.
Sure to appeal to fans of Call the Midwife and Our Girl, this is a wonderfully nostalgic trio of real-life reads that shines a light on what life was like for the women left at home during the wartime era and beyond.
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The touching GI Brides takes a look at four girls who left their homeland to follow their new husbands across the Atlantic shortly after the end of the War. Not only did these girls - Gwendolyn, Rae, Margaret and Sylvia - have to cope with a new culture, they also had to get to know their husbands outside of the army... But after the Blitz, they were sure they could cope with anything and this book reveals how much fun they had!
The Sweethearts is about those women who worked at the Rowntree's factory in York from the 1930s right the way through to the 1980s and how they kept the treats coming, no matter what was thrown at them... The Sugar Girls follows the women who worked at Tate & Lyle's factory in the East End during the Second World War - they're the ones who kept Britain sweet during the time of rations.
Fizzing with suspense from the first page to the last, our collection of David Downing books is an outstanding set of three post-war fiction titles that follow the exploits of British spy John Russell.
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Fast forward four years since Russell left his son, Paul, and lover Effi Koenen in volatile Berlin and Potsdam Station sees Russell return to Germany to find out if they're alive and to protect them from the advancing Red Army. But his plan is faltered by the Soviets, who find a use for him that has him parachuting into Berlin behind the German lines.
Forced into becoming a double agent for the Soviets in Lehrter Station; and embarking on the most dangerous task of his life in Masaryk Station our David Downing Collection is convincing, compelling and captivating - just perfect for fans of C. J. Sansom and Robert Harris.
This book contains books 3-6 of David Downing's John Russell series.
Focusing on the treatment of casualties during World War I, our Pat Barker Collection is a powerful, harrowing examination of the insanity of conflict. Here, we are delighted to bring you her complete Regeneration series for less than the RRP of just one book - we're sure once you pick them up, you won't be able to put them down.
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In Regeneration, the first book in the series, a decorated English officer is sent to a military hospital after publicly declaring he will no longer fight. Combining real-life and fictional characters, and issues of class and politics, the book effortlessly weaves heart-rending prose and expert plotting.
Completing this trio of poignant wartime novels are The Eye in the Door and the Man Booker Prize-winning The Ghost Road.
An ideal set of books for anyone who is interested in powerful writing, historical fiction and military history, our three-book Pat Barker Collection is shocking, eye-opening and completely immersive.
This collection contains books 1-3 in Pat Barker's Regeneration series.
M.C. Scott's Rome books brilliantly bring to life a fascinating and popular historical era. The three novels included in this collection are all extremely gripping and capture the passion, tension and intrigue of the time.
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The Emperor's Spy finds a mysterious prophecy foretelling that Rome will burn, and only one man can save it, while in The Coming of the King Sebastos Pantera, known to his enemies as the Leopard, must find the most dangerous man in the empire and bring him to justice. The Eagle of the Twelfth tells the story of Demalion and his legionnaires as they go undercover into Jerusalem to recover their pride...
Likely to appeal to fans of Simon Scarrow and Conn Iggulden, this exciting and captivating historical fiction set is sure to keep you hooked!
MZK2 11 years +11 years +
Now a major motion picture starring Geoffrey Rush and Emily Watson, The Book Thief is a moving and breathtaking novel that young readers and adults will both be sure to devour.
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Set in 1939 Nazi Germany, the country holds its breath, waiting on the brink of war. Young Liesel's life is changed forever as she stands by her brother's grave and picks up The Gravedigger's Handbook from the snow, committing her first act of book thievery and changing her life forever... So follows her love affair with books in an increasingly dangerous time. When her foster family hide a Jewish boy in their basement her world is both opened up and shut down, but the power of words and the ability for books to feed the soul is explored and enjoyed by both youngsters.
Superbly crafted and incredibly moving, children aged 11+ simply must read this award-winning tale which has proved to be one of the most enduring and respected of recent times.
WWI Centenary Edition. With a new introduction from the author. A novel of overwhelming emotional power, Birdsong is a story of love, death, sex and survival. Stephen Wraysford, a young Englishman, arrives in Amiens in northern France in 1910 to stay with the Azaire family, and falls in love with unhappily married Isabelle. But, with the world on the brink of war, the relationship falters, and Stephen volunteers to fight on the Western Front. His love for Isabelle forever engraved on his heart, he experiences the unprecedented horrors of that conflict - from which neither he nor any reader of this book can emerge unchanged.
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A mother's struggle to protect her family and escape Nazi persecution in World War Two Germany. Germany 1937: Fear and betrayal stalk the streets. People disappear. Persecution of the Jews has become a national pastime. When Ruth Friedman's husband is arrested by the SS, she is left to fend for herself and her four children. She alone stands as their shield against the Nazis. But where can she go? Where will her family be safe? Ruth must overcome the indifference, hatred and cruelty that surrounds her as she and her family race to escape the advancing Nazi army's final solution...
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The master of historical fiction presents the iconic story of King Alfred and the making of a nation. Alfred, the great king, is said to be dying. Rivals for his succession are poised to tear the kingdom apart. The country Alfred has worked thirty years to build is about to disintegrate. Uhtred, the King's warrior, Viking born but Saxon bred, wants more than anything else to go and fight to reclaim his stolen Northumbrian inheritance. But he knows that if he deserts the King's cause, Alfred's dream -- and indeed the very future of the English nation -- will very likely vanish. Death of Kings is an outstanding novel by a master storyteller of how England was made -- and very nearly lost.
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Historical fiction master Conn Iggulden retells the gripping story of the English civil war in his new Wars of the Roses series. King Henry V - the great Lion of England - is long dead. In 1437 his gentle son comes of age and takes the throne. Frail in body and mind, King Henry VI is dependent on his supporters to run his kingdom. Richard, Duke of York, however, believes that without a strong king England will fall. His fears seem justified as English power comes under threat from France, and discontent and rebellion spread at home. On the counsel of his advisers, Henry marries the young princess Margaret of Anjou, in order to forge an alliance with France - but is it too late? As the storm clouds gather, King Henry and his queen are besieged abroad and at home. Who can save the throne? Who will save the kingdom? The Wars of the Roses series will be a benchmark for historical fiction, showcasing Conn Iggulden at his finest. Praise for Conn Iggulden: "This is energetic, competent stuff; Iggulden knows his material and his audience". (Independent). "Iggulden is in a class of his own when it comes to epic, historical fiction". (Daily Mirror). "Iggulden ...tells an absolutely cracking story". (The Times). Conn Iggulden is the author of the best-selling Emperor and Conqueror historical fiction series and also the co-author (with his brother Hal), of 2006's British Book of The Year, The Dangerous Book for Boys. Born in London, Conn Iggulden read English at London University and worked as a teacher for seven years before becoming a full time writer. He is married and lives in Hertfordshire with his wife and children.
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The new novel in Bernard Cornwell's number one bestselling series The Warrior Chronicles, on the making of England and the fate of his great hero, Uhtred of Bebbanburg. In the battle for power, there can be only one ruler. The ruler of Mercia is dying, leaving no apparent heir. His wife is a born leader, but no woman has ever ruled over an English kingdom. And she is without her greatest warrior and champion, Uhtred of Bebbanburg. An empty throne leaves the kingdom exposed to rival West Saxons and to the Vikings, who are on a bloody rampage once more. A hero is needed, a hero who has been in battle all his life, who can destroy the double threat to Mercia. A hero who will ultimately decide the fate of a nation...
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'A master of the genre' The Times The ninth novel in the thrilling Empire sequence leads Centurion Marcus Aquila and the Tungrians to the battlefield that was one of Rome's most disastrous defeats. The Tungrians have no sooner returned to Rome than they find themselves tasked with a very different mission to their desperate exploits in Parthia. Ordered to cross the river Rhenus into barbarian Germany and capture a tribal priestess who may be the most dangerous person on the empire's northern border, they are soon subject to the machinations of an old enemy who will stop at nothing to sabotage their plans before they have even set foot on the river's eastern bank. But after their Roman enemy is neutralised they face a challenge greater still. With two of the Bructeri tribe's greatest treasures in their hands they must regain Roman territory by crossing the unforgiving wilderness that was the graveyard of Roman imperial strategy two hundred years before. And capture by the Bructeri's vengeful chieftain and his warband can only end in one way - a horrific sacrificial death on the tribe's altar of blood.
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A Sunday Times bestseller. Simon Scarrow's veteran Roman soldier heroes face a cunning and relentless enemy in BRITANNIA, the unforgettable fourteenth novel in the bestselling Eagles of the Empire series. Roman Britain, AD 52. The western tribes prepare to make a stand. But can they match the discipline and courage of the legionaries? Wounded Centurion Macro remains behind in charge of the fort as Prefect Cato leads an invasion deep into the hills. Cato's mission: to cement Rome's triumph over the natives by crushing the Druid stronghold. But with winter drawing in, the terrain is barely passable through icy rain and snowstorms. When Macro's patrols report that the natives in the vicinity of the garrison are thinning out, a terrible suspicion takes shape in the battle-scarred soldier's mind. Has the acting Governor, Legate Quintatus, underestimated the enemy? If there is a sophisticated and deadly plan afoot, it's Cato and his men who will pay the price...Includes maps, chart and author Q&A.
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Explosive, subversive, wild and funny, 50 years on the novel's strength is undiminished. Reading Joseph Heller's classic satire is nothing less than a rite of passage. Set in the closing months of World War II in an American bomber squadron off the coast of Italy, "Catch-22" is the story of a bombardier named Yossarian who is frantic and furious because thousands of people he has never even met keep trying to kill him. Joseph Heller's bestselling novel is a hilarious and tragic satire on military madness, and the tale of one man's efforts to survive it.
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This book was the winner of the Man Booker Prize 2014. Forever after, there were for them only two sorts of men: the men who were on the Line, and the rest of humanity, who were not. In the despair of a Japanese POW camp on the Burma Death Railway, surgeon Dorrigo Evans is haunted by his love affair with his uncle's young wife two years earlier. Struggling to save the men under his command from starvation, from cholera, from beatings, he receives a letter that will change his life forever. This is a story about the many forms of love and death, of war and truth, as one man comes of age, propsers, only to discover all that he has lost.
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1945: a lost German bomber crashes on the Vatnajokull glacier in Iceland. Inexplicably, in the midst of World War Two, there are both German and American officers on board. One of the senior German officers claims that their best chance of survival is to try to walk to the nearest farm and sets off, a briefcase handcuffed to his wrist. 1999, mid-winter, and the US Army is secretively trying to remove an aeroplane from the Vatnajokull glacier. By coincidence two young Icelanders become involved - but will pay with their lives. Before they are captured, one of the two contacts his sister, Kristin, who will not rest until she discovers the truth of her brother's fate. Her pursuit puts her in great danger, leading her on a long and hazardous journey in search of the key to the riddle about Operation Napoleon.
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In the spring of 1966, while the war in Vietnam was still popular, the US military decided to reactivate the 9th Infantry Division as part of the military build-up. Across the nation, farm boys from the Midwest, surfers from California and city-slickers from Cleveland opened their mail to find greetings from Uncle Sam. Most American soldiers of the Vietnam era trickled into the war zone as individual replacements for men who had become casualties or had rotated home. Charlie Company was different as part of the only division raised, drafted and trained for service. From draft to the battlefields of South Vietnam, this is the unvarnished truth from the fear of death to the chaos of battle, told almost entirely through the recollections of the men themselves. This is their story, the story of young draftees who had done everything that their nation had asked of them and had received so little in return - lost faces of a distant war.
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Lieutenant-Colonel Sharpe, sidelined on the Royal staff, magnificently siezes command at the final moment of the great victory. It is 1815. Sharpe is serving on the personal staff of the Prince of Orange, who refuses to listen to Sharpe,s reports of an enormous army, led by Napoleon, marching towards them. The Battle of Waterloo commences and it seems as if Sharpe must stand by and watch the grandest scale of military folly. But at the height of battle, as victory seems impossible, Sharpe takes command and the most hard-fought and bloody battle of his career becomes his most magnificent triumph. Soldier, hero, rogue - Sharpe is the man you always want on your side. Born in poverty, he joined the army to escape jail and climbed the ranks by sheer brutal courage. He knows no other family than the regiment of the 95th Rifles whose green jacket he proudly wears.
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A sequence of stories about the Vietnam War, this book also has the unity of a novel, with recurring characters and interwoven strands of plot and theme. It aims to summarize America's involvement in Vietnam, and her coming to terms with that experience in the years that followed.
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The year is 1809, and the recruiting sergeants are hard at work, as the British army gathers strength for the next phase of the campaign against Bonaparte on the Spanish Peninsula. Captain Billy Pringle of the 106th Foot, however, has a somewhat more urgent reason to leave the country: having become embroiled in an ill-advised duel with a lieutenant in the 14th Light Dragoons, a posting to Spain would avoid any awkwardness for the regiment. Along with his friend Lieutenant Williams - whose sister Kitty was the cause of the duel - and the doughty veteran Sergeant Dobson, Pringle takes on the task of training Spanish troops to stand alongside their British allies. But what seems at first like easy duty soon turns into a desperate fight for survival as they find themselves besieged in the strategic fortress of Cuidad Rodrigo. For Bonaparte, taking the fortress will be the first step towards pushing the British back to the sea, and the task is entrusted to one of his most daring and successful generals, Marshal Ney. And Ney in his turn has found the perfect officer to lead the assault, a man not only desperate for advancement but also thirsting for revenge - a man whom Williams knows only too well.
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Ben Tyson is a good man, a brilliant corporate executive, an honest handsome family man, admired by men and desired by women. But sixteen years ago Ben Tyson was a lieutenant in Vietnam. There, in 1968, the men under his command committed a murderous atrocity - and together swore never to tell the world what they had done. Now the press, army justice and the events he tried to forget have caught up with Ben Tyson. His family, his career and his personal sense of honour hang in the balance.
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The year is 1808, and Hamish Williams is a 'gentlemen volunteer' in the 106th regiment of foot, a man serving with the ranks but living with the officers, and uncomfortable in both worlds: looked down on by those with the money or influence to buy their rank, and distrusted by the common soldiers who know he is not one of them. But Williams is determined to prove by deeds alone that he is a man worthy of advancement, and when the 106th embarks for Portugal to begin what will become known as the Peninsula War against Napoleon, he knows his chance of glory is at hand. Soon he is receiving a sharp lesson in the realities of war, as the 106th undergoes a bloody baptism at the hands of the French - and he realises that his single-minded devotion to honour may not, after all, be the quickest route to promotion. Combining the vivid detail of a master historian with the engaging characters and pulsating action of a natural storyteller, TRUE SOLDIER GENTLEMAN is the first volume in what promises to be a classic series.
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Sennheiser teams with Google for Ingress headphones
There's a game called Ingress out there, ladies and gentlemen, and it's Google's game. They promote it at every event, they play it, they live it. It's a game you play with your smartphone, GPS, and a lot of patience. The folks at Sennheiser have teamed up with Google to bring an added dimension of addiction to those that love the game with a couple of pairs of headphones. One for the "Enlightened" side of the game, the other from the "Resistance."


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A history of the
Girl Scouts
October 31, 1860: Juliette Magill Kinzie Gordon, nicknamed "Daisy," is born in Savannah, Georgia.
1882: Daisy goes on her first trip to Europe.
1886: Daisy marries William Mackay Low, "Billow," in Savannah.
1887: Daisy and Billow move to England.
1905: Billow dies.
1911: Daisy meets Sir Robert Baden-Powell, founder of the Boy Scouts.
1912: Daisy gathers the first American Girl Scouts for a meeting in Savannah.
1915: Daisy is named as the first president of the Girl Scouts.
1927: On January 17, in Savannah, Daisy dies from breast cancer. She was 66.
1948: A United States postage stamp featuring Daisy's picture is issued. (It's shown on Page 1.)
1953: The Girl Scouts buy and restore Daisy's childhood home.
1979: Daisy is inducted into the National Women's Hall of Fame in Seneca Falls, New York.
2005: Daisy is honored with a medallion on "The Extra Mile Points of Light Volunteer Pathway" near the White House.
2012: Girl Scouts of the USA celebrates 100 years since its founding by Juliette Gordon Low.