Conditional Wwox Deletion in Mouse Mammary Gland by Means of Two Cre Recombinase Approaches
Published May 4, 2012

Brent W Ferguson
Department of Molecular Carcinogenesis
The University of Texas M.D. Anderson Cancer Center
SmithvilleTexasUnited States of America

Xinsheng Gao
Department of Molecular Carcinogenesis
The University of Texas M.D. Anderson Cancer Center
SmithvilleTexasUnited States of America

Hyunsuk Kil
Department of Molecular Carcinogenesis
The University of Texas M.D. Anderson Cancer Center
SmithvilleTexasUnited States of America

Jaeho Lee
Department of Molecular Carcinogenesis
The University of Texas M.D. Anderson Cancer Center
SmithvilleTexasUnited States of America

Fernando Benavides
Department of Molecular Carcinogenesis
The University of Texas M.D. Anderson Cancer Center
SmithvilleTexasUnited States of America

Martin C Abba
Facultad de Medicina
CINIBA
Universidad Nacional de La Plata
La PlataArgentina

C Marcelo Aldaz email@example.com
Department of Molecular Carcinogenesis
The University of Texas M.D. Anderson Cancer Center
SmithvilleTexasUnited States of America

Conditional Wwox Deletion in Mouse Mammary Gland by Means of Two Cre Recombinase Approaches
Published May 4, 2012Received February 7, 2012; Accepted April 3, 2012;Citation: Ferguson BW, Gao X, Kil H, Lee J, Benavides F, et al. (2012) Conditional Wwox Deletion in Mouse Mammary Gland by Means of Two Cre Recombinase Approaches. PLoS ONE 7(5): e36618. Editor: Robert Oshima, Sanford Burnham Medical Research Institute, United States of America http://cancer.gov/) to BWF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. *
Loss of WWOX expression has been reported in many different cancers including breast cancer. Elucidating the function of this gene in adult tissues has not been possible with full Wwox knockout models. Here we characterize the first conditional models of Wwox ablation in mouse mammary epithelium utilizing two transgenic lines expressing Cre recombinase, keratin 5-Cre (BK5-Cre) and MMTV-Cre. In the BK5-Cre model we observed very efficient Wwox ablation in KO mammary glands. However, BK5-Cre Wwox KO animals die prematurely for unknown reasons. In the MMTV-Cre model we observed significant ablation of Wwox in mammary epithelium with no effect on survival. In both of these models we found that Wwox deletion resulted in impaired mammary branching morphogenesis. We demonstrate that loss of Wwox is not carcinogenic in our KO models. Furthermore, no evidence of increase proliferation or development of premalignant lesions was observed. In none of the models did loss of a single Wwox allele (i.e. haploinsufficiency) have any observable phenotypic effect in mammary gland. To better understand the function of Wwox in the mammary gland, transcriptome profiling was performed. We observed that Wwox ablation results in the deregulation of genes involved in various cellular processes. We found that expression of the non-canonical Wnt ligand, Wnt5a, was significantly upregulated in Wwox KO mammary epithelium. Interestingly, we also determined that components of the Jak/Stat3 signaling pathway were upregulated in KO mice and this correlated with a very robust increase in phospho-Stat3 signaling, which warrants further testing. Even though the loss of Wwox expression in breast and other cancers is very well documented, our findings suggest that Wwox does not act as a classical tumor suppressor as previously thought.

Introduction

The WW domain-containing oxidoreductase (WWOX) gene was identified as a potential tumor suppressor gene mapping to chromosomal region 16q23 [1,2]. The WWOX gene spans .1.1 Mb and overlaps the FRA16D common chromosomal fragile site [1,2,3]. Disruptions of the WWOX locus by hemi, homozygous deletions and other rearrangements have been reported in various human malignancies including breast, prostate, ovarian, lung, liver cancer and multiple myeloma [1,4,5]. WWOX is highly conserved throughout evolution and in mouse the Wwox gene is 93% identical to its human counterpart. Interestingly the mouse Wwox gene also co-localizes with the murine chromosomal fragile site Fra8E1 [6].

Significant research has been performed to elucidate the normal and putative tumor-suppressive roles of WWOX. The WWOX protein has a molecular mass of 46 kD with two functional motifs consisting of two N-terminal WW domains and a short-chain dehydrogenase (SDR) domain [1,7]. The WW domains of WWOX have been found to be essential for protein-protein interactions with proteins harboring PPXY motifs [8] and the substrate/s for the WWOX NADH/NADPH oxidoreductase enzymatic domain are yet to be identified. Several potential protein partners have been identified and include regulators of cell growth and apoptosis as well as various transcription factors [8,9,10,11,12,13]. The putative role of WWOX as a tumor suppressor has been suggested by various studies observing suppressed in vivo tumorigenicity of human cancer cells when WWOX was ectopically overexpressed [14,15,16,17].

Our group, as well as others, have generated mouse models of Wwox knockdown to gain a better understanding of the in vivo functions of Wwox in development and cancer. Aqeilan et al. generated a full knockout (KO) mouse model via targeted disruption of the gene. Even though all mice die by 3-4 weeks these authors reported the development of periostal osteosarcomas but only in 3 of 11 pups analyzed [18]. Our group generated and characterized mice harboring a conditional Wwox allele in which exon 1 was flanked by loxP sites [19]. Upon breeding these mice with EIIA-Cre transgenic mice, we generated full Wwox KO mice and observed that these mice displayed severe metabolic defects, growth retardation and all mice died by 3 weeks of age as previously observed [19], however in contrast with the findings of Aqeilan and coworkers no evidence of spontaneous neoplasia was detected in these short-lived Wwox KO mice [19].

We are interested in understanding the normal and putative tumor-suppressive functions of WWOX in more detail. Since we and others observed consistent and significant loss of WWOX expression in breast cancer [20,21,22,23] we focused first in the mammary gland. Here we report the characterization of conditional ablation of Wwox in mouse mammary gland using two approaches, we utilized transgenic lines with Cre recombinase under control of a Keratin 5 promoter (BK5-Cre) and also under the MMTV promoter (MMTV-Cre) in order to inactive Wwox in the mammary epithelium.

Results

Assessment of two Wwox conditional KO models in mouse mammary epithelium

To determine the role of Wwox in the murine mammary gland, Wwox flox/flox mice were mated with mice carrying a transgene expressing Cre recombinase resulting in the conditional deletion of the Wwox gene in mammary epithelium. To this end we tested two independent models of Cre driven recombination. The first conditional KO model had Cre recombinase expression driven by the bovine keratin 5 promoter (BK5-Cre). The BK5 promoter is activated in utero (E13.5) in epithelial precursor cells [24]. In the second model, the MMTV promoter drove Cre expression (MMTV-Cre). The MMTV promoter in the transgenic Line D is expressed in various tissues and is maximally turned on in mammary gland epithelium at approximately 22 days of age [25].

To examine Wwox protein expression and the efficacy of Wwox ablation in each of these models we analyzed histological sections of mammary glands from female mice stained with Wwox antibody. We observed high levels of Wwox expression in the cytoplasm of mammary epithelial cells of 8 week virgin BK5 Wwox WT control mice and this expression was largely absent in BK5 KO mice (Figures 1 A and B). This loss of Wwox expression was also clear in mammary glands from pregnant (P18.5 days) Wwox KO mice even after the burst of epithelial proliferation associated with pregnancy ( Figures 1 C and D). In the MMTV-Cre model we found that Wwox ablation was very effective as well (Figures 1 E and F). We observed however isolated patches of epithelial structures expressing Wwox indicative of some mosaicism but only in some mice. This heterogeneity in the MMTV Cre model is not uncommon with Cre systems and is likely due to the mosaic nature of Cre expression and perhaps incomplete recombination affecting some cells in some mice. However, this mosaicism does not result in repopulation of the mammary gland by Wwoxpositive epithelium as discussed in a follow up section.

We further analyzed the extent of Wwox deletion in the BK5-Cre model at the RNA level by real-time PCR. We observed that Wwox expression was dramatically decreased in both 8 week virgin and P18.5 Wwox KO mammary epithelial organoids when compared to their WT counterparts ( Figure 2). These results demonstrate that Wwox deletion by use of the BK5-Cre and MMTV-Cre transgenic lines is very effective in mouse mammary epithelium.

Long-term follow up experiments BK5-Cre model

In order to determine the effects of long-term Wwox ablation in the mammary gland and to test whether lack of Wwox expression was tumorigenic we set up standard survival experiments. BK5-Cre + ; Wwox flox/flox males and females survived weaning, were fertile and females were able to lactate. However, inexplicably, BK5 KO mice died prematurely at an early age. The youngest animal died at 68 days of age and no BK5 KO mouse survived longer that 117 days of age (0% survival, n = 22). This premature death was independent of gender as 9 females and 13 males were included in the study. BK5-Cre (2) ; Wwox flox/flox (WT), mice exhibited 100% survival over the same observation period. It is also important to note that loss of one Wwox allele in the BK5-Cre (+) ; Wwox +/flox heterozygous mice had no effect on survival (100% survival, n = 41, Figure S1). Numerous groups of experimental animals were generated independent of the described survival study with the same results. We performed comprehensive histopathological, hematological and blood chemistry analyses on these BK5-Cre Wwox KO mice before dead and could not determine a reason for the relatively sudden deaths.

We observed no tumor formation in any of the BK5 KO mammary glands during their relatively short lifespan (mean = 115 days). Importantly, none of the BK5-Cre (+) ; Wwox +/flox heterozygous mice develop tumors even after over a year of follow up, indicating that lack of one Wwox allele (i.e. equivalent to haploinsufficiency) is not tumorigenic in the mouse mammary gland.

Since Keratin 5 is highly expressed in cells of the basal layer of multiple epithelia including the skin, we carefully inspected the epidermis of BK5 KO mice. We confirmed lack of Wwox expression in epidermis of BK5 KO mice by means of immunohistochemistry (data not shown). We observed no obvious macro or microscopic abnormal skin phenotype. Furthermore, BK5 KO mice displayed the normal bilayer architecture of mouse skin with  24 week MMTV Wwox KO. Rabbit anti-Wwox [14] antibody was used at a 1:50 dilution and counterstained with hematoxylin. All images were obtained at the same 106 magnification. doi:10.1371/journal.pone.0036618.g001 no signs of hyperplasia or abnormalities in differentiation. No skin premalignant or tumorigenic lesions spontaneously developed in either BK5 KO or heterozygous mice.

Histopathological evaluation of mammary gland tissue sections from multiple BK5 KO and Wwox +/flox mice at various stages of development, revealed no signs of malignancy or premalignant changes. As can be observed in Figure 1 B no evidence of hyperplasia is noted. Similarly, no evidence of malignancy or hyperplastic nodules were observed in any of the whole mount preparations obtained from BK5 KO mice.

Keratin 5 immunostaining on BK5 KO mammary gland revealed no obvious abnormalities in differentiation ( Figure S2). We also analyzed the effect of Wwox ablation on proliferation in the mammary gland via immunohistochemical staining for Ki-67 and found no statistical difference between WT and KO mice in the BK5-Cre model (data not shown). Importantly, we also found no difference in ERa or PR expression between the BK5 Wwox WT and KO groups as determined by immunohistochemical staining on mammary gland tissue sections from 8 week and 12 week virgin female mice (data not shown).

In order to extend the observation period to monitor the effects of Wwox ablation in mammary epithelium of the BK5 KO model, we performed transplantation experiments of mammary epithelium from 12 week old BK5 KO mice into the cleared mammary fat pads of 4-5 week old virgin SCID female mice (n = 10). We followed the SCID recipient mice for 9 additional months and did not observe tumor formation in any of the recipient mice. At the end of the observation period transplanted mammary fat pads were dissected and prepared for whole mounts. The obtained mammary pad whole mounts were evaluated after carmine alum staining confirming the successful grafting of the donor epithelium in 100% of the cases. We did not observe the development of any premalignant or malignant lesions in these whole mount preparations (data not shown).

Long-term follow up experiments MMTV-Cre model

The MMTV-Cre line D expresses the Cre recombinase in luminal and myoepithelial cells of the mammary gland and is significantly expressed at approximately 22 days of age [25]. In contrast with the BK5-Cre KO model, we observed that Wwox deletion by means of the MMTV-Cre system had no effect in survival and natural aging. We followed 60 MMTV-Cre(+); Wwox flox/flox , i.e. KO mice (30 females, 30 males) and 23 MMTV-Cre(2); Wwox flox/flox , i.e. WT mice (13 females, 10 males) for up to 1.4 years and observed no statistical difference in survival between the two groups. Importantly, and like with the BK5-Cre model, when whole mounts or histological sections of mammary glands were analyzed we found no evidence of hyperplasia, premalignant or malignant lesions ( Figure 1F). We also observed no evidence of tumor formation in any other tissue known to be affected by MMTV-Cre such as the salivary gland [25].

Given the moderate to mild mosaicism in Wwox expression detected in some MMTV-Cre KO mice, we investigated whether Wwox-positive epithelial cells could gain an advantage over the Wwox-negative epithelium and repopulate the mammary gland later in life. To this end we analyzed Wwox expression in the mammary glands of WT (n = 8) and KO (n = 17) mice when they reached 1.4 years of age at the end of the survival study. We found as expected that all Wwox WT mammary glands were still strongly positive for Wwox expression and we observed that over half of the Wwox KO glands were completely negative (n = 9) for Wwox expression indicating effective deletion of Wwox while the remaining samples (n = 8) showed some mosaicism with areas displaying some epithelial structures positive and other negative for Wwox expression, indicating that even though the mosaicism remained the Wwox expressing cells do not overtake the mammary gland.

Wwox deletion results in aberrant mammary branching morphogenesis

By simple microscopic inspection of the mammary whole mounts obtained from the BK5 Wwox KO and MMTV Wwox KO mice we observed a significant impairment in branching development when comparing with the WT counterparts. In some cases the impairment in mammary fat pad ductal invasion and expansion was very dramatic and extreme developing just a rudimentary mammary epithelium or just a few ductal structures with little or no branching (Figures 3 A-C).

In order to quantitate these observations we performed morphometric determinations of mammary branching. To this end whole mounts of the 4 th inguinal mammary glands were collected from virgin female mice at 12 weeks of age from BK5 Cre(2) WT, BK5 Cre(+) WT and BK5 KO. We observed some impairment in branching morphogenesis in the control glands expressing Cre recombinase when compared with the Cre negative counterparts. However we observed a statistical significant decrease in branching in the mammary glands from the BK5 Wwox KO mice when compared with either of the controls (Figures 3 A, B, and D).

Importantly, the impaired branching observed in the BK5 KO mammary gland did not impede the mammary epithelium to reach functional maturity and differentiation during pregnancy. Furthermore, BK5 Wwox KO mice were able to lactate and expressed similar levels of the differentiation markers Wap and bcasein as determined by qRT-PCR (data not shown).

In the MMTV-Cre model of Wwox deletion we also observed a similar phenomenon in reduced branching ( Figure 3C). However such phenotype was more heterogeneous than with the BK5-Cre model. The branching phenotype in MMTV Wwox KO mice ranged from very severe (Figure 3 C) to more similar to wild-type. We did not detect statistically significant differences at 12 weeks of age given the large standard deviations observed in MMTV Wwox KO mice (data not shown). However, analyses from older MMTV-Cre females (24 weeks of age) detected significant differences in branching (6.8+/20.32 vs. 3.8+/21.33 branches/mm, p = 0.017, Figures 3 C and E).

Global gene expression changes in Wwox ablated mammary epithelium

To determine the effect of Wwox ablation on global gene expression in mammary epithelium we performed gene expression microarray analyses on RNA obtained from 8 week old BK5 Wwox KO and WT mammary epithelium (n = 3 WT mice and 3 KO mice). We decided to pursue these studies with the BK5-Cre model given the uniformity and efficiency in Wwox ablation. The statistical analysis of the Affymetrix gene chip expression profiling data identified 913 probes differentially expressed between Wwox KO and Wwox WT mammary gland epithelium (p,0.01; 2 fold changes). Among the 913 probes, 777 were up-modulated and 136 were down-modulated in the mammary gland epithelium derived from the Wwox KO mice model ( Figure 4A and B; Table S1). We used the DAVID resource for automated annotation and enrichment analysis of the differentially expressed genes based on GO database. In addition, we employed REVIGO resource for summarization and visualization of the significant GO term semantic similarities (p,0.025). Among the statistically significant over-represented categories under Biological Processes, we found the Wnt signaling pathway, skeletal system development/bone morphogenesis, and tissue remodeling related genes (Figure 4 C). In addition, categories of genes found in cell migration and adhesion were also highly enriched related genes in the Wwox-KO mammary gland expression profile.

Validation of gene expression changes

One of the most significantly upregulated genes in Wwox-KO mammary epithelium was the non-canonical Wnt pathway ligand Wnt5a (fold change = 3.01, p = 0.000057). Wnt5a is expressed during all stages of mammary gland development except during lactation [26]. We sought to validate the findings of our microarray by performing semi-quantitative RT-PCR on RNA from the mammary gland epithelial organoids of three 8 week old WT mice compared to three Wwox-KO mice ( Figure 5, lanes 1-3 and 7-9). Wnt5a expression was observed to be variable in unsynchronized mice; mammary epithelial samples from two WT mice displayed very low levels of Wnt5a expression while one displayed high Wnt5a expression. We observed that all Wwox-KO mammary epithelial samples exhibited upregulation of Wnt5a. We then tested this expression in the pregnancy synchronized epithelium of P18.5 mice and found that 2 out of 3 WT mammary glands displayed low levels of Wnt5a expression while all Wwox-KO mammary glands tested exhibited increased levels of Wnt5a mRNA ( Figure 5, lanes 4-6 and 10-12).

We then sought to determine possible mechanisms by which Wnt5a expression could be upregulated in Wwox-KO mammary gland. IL-6 induced Jak/Stat3 signaling has been shown to activate Wnt5a expression in many different cell types [27,28]. From our microarray analysis we identified significant upregulation of several genes related with the Jak/Stat3 signaling pathway (see Table S1). For instance, IL6st, the coreceptor and signal transducer of the pathway, was upregulated 4.17 fold (p = 0.005), Jak1 was upregulated 3.04 fold (p = 0.003) and Stat3 was upregulated 2.4 fold (p = 0.01). To determine whether Stat3 was activated (i.e. phosphorylated) we analyzed phospho-Stat3 expression by means of immunohistochemistry in mammary gland samples from BK5 Wwox WT (n = 3) and KO (n = 3) mammary glands. We found that Wwox KO mammary epithelium had a significantly higher number of cells that were positive for strong nuclear phospho-Stat3 staining (p,0.003) (Figures 6 A-C). We also compared levels of phospho-Stat3 by means of Western blotting in BK5-Cre WT and KO epithelial organoids at p18.5, we indeed found a significant increase in phospho-Stat3 expression in the KO samples ( Figure 6D).

In the canonical Wnt pathway we found that several genes were significantly upregulated in Wwox KO mammary epithelium. This pathway was also highlighted by the GO analysis of the gene expression data ( Figure 4C). Gsk3b was increased 6.65 fold (p = 0.0044). In addition, we identified over-expression of the Lrp6 coreceptor (1.8 fold; p = 0.0004) and the signal transducer Dishevelled (Dvl1) (1.8 fold; p = 0.0033) at a less stringent statistical cutoff (under 2 fold changes). The b-catenin gene was upregulated 3.85 fold (p = 0.0007) in our microarray. To determine whether the noted upregulation of b-catenin mRNA and other canonical pathway components resulted in an increase of nuclear localized b-catenin protein we performed immunohistochemical analysis on mammary gland sections from 12 week old virgin BK5-Cre mice. We found that all protein was localized to cytoplasm and cell membrane, no nuclear localization was noted and no differences were observed between BK5 WT and KO mammary samples, indicating that likely most b-catenin is degraded in the cytoplasmic compartment and does not translate in hyperactivation of said pathway. This is consistent with the findings that no classical target genes of the canonical Wnt pathway were observed upregulated in KO mammary glands (e.g. Cyclin D1, Myc, Fgf20) and no change in proliferation was observed.

Discussion

Previous mouse Wwox KO models have shown that deletion of this gene dramatically affects the lifespan of the animal, all mice die by 3-4 weeks of age thus making observations in adult tissues impossible [18,19]. Here we present the first report on the effects of conditional Wwox deletion in developing and mature mouse mammary epithelium. One of the primary goals of this study was to determine whether deleting Wwox in the mammary gland leads to tumor formation. Importantly, we found no evidence of tumor formation in the mammary gland or any other tissue by either of the two conditional KO approaches utilized, even if these mice were monitored up to 1.4 years of age. This strongly suggests that Wwox does not behave as a classical highly penetrant tumor suppressor gene in mammary gland epithelium as previously thought [4]. Our studies clearly indicate that loss of Wwox expression is not sufficient for tumor formation. Furthermore, our findings are in clear disagreement with previous speculation suggesting that Wwox haploinsufficiency is tumorigenic [18,29,30,31]. Importantly, Wwox ablation did not lead to the development of hyperplasia or preneoplastic lesions and no increase in cell proliferation was observed in mammary gland epithelium. This suggests that Wwox loss of expression is perhaps of more relevance during the progression stages of tumorigenesis rather than in the initiation of neoplasia. In fact, we did find that loss of Wwox in the mammary gland is associated with an increase in the expression of several genes associated with tumor progression.

In previous studies, Abdeen et al reported increased susceptibility to mammary carcinogenesis in Wwox+/2mice that were backcrossed onto the mammary tumor-susceptible C3H genetic background. Thus, an effect of Wwox in tumor initiation cannot be ruled out at this point. There is also a possibility that genetic background may play a role in masking some mammary phenotypic effects of Wwox ablation since our studies were performed in a mixed 129SV/C57Bl/6 genetic background. However, it is unlikely that we would not see any signs of mammary cancer predisposition as described throughout this report.

We further observed that Wwox ablation results in a significant impairment in mammary gland ductal branching in virgin mice. Perhaps the lack of Wwox expression affects the ability of epithelial cells to invade the mammary fat pad during development. Furthermore several genes associated with tissue remodeling and extracellular matrix interactions were observed deregulated. For instance, Timp2 and Timp3 were both found significantly upregulated in Wwox KO epithelium (Table S1).

However, the observed defects in mammary gland morphogenesis did not inhibit normal functional differentiation since both BK5 and MMTV Wwox KO mice are able to produce mature mammary epithelial gland structures during pregnancy leading to milk production and normal lactation.

In our gene expression studies we found that the Wnt5a gene is significantly upregulated in Wwox-ablated mammary epithelium. The Wnt5a ligand primarily signals through the planar cell polarity pathway but has the potential to activate the canonical Wnt signaling pathway as well depending on which set of coreceptors pair with the Frizzled receptor upon Wnt5a binding [32]. High levels of Wnt5a expression have been associated with osteosarcoma [33], prostate carcinoma [34] and metastatic melanoma [35]. Interestingly in normal tissue such as the developing mammary gland high levels of Wnt5a are associated with decreased cell proliferation and delayed development [36]. Furthermore, Wnt5a over-expression has been shown to lead to developing a very similar phenotype to what we observed in Wwox KO mammary epithelium. Wnt5a overexpression inhibits ductal extension and lateral branching while Wnt5a 2/2 mammary tissue exhibits an accelerated developmental capacity compared with wild-type tissue [36].

A number of different cell signaling pathways have been shown to activate transcription of the Wnt5a gene such as TGFb, TNFa, Hedgehog, and IL-6/Jak/Stat3 (reviewed in [37]). We chose to focus on the Jak/Stat3 pathway not only because of its association with Wnt5a expression but also because several transcripts of said pathway were detected upregulated in Wwox KO mammary epithelium.

Mammalian Wnt5a is known to be a Stat3-target gene due to tandem Stat-binding sites found in a region of intron 4 of the gene [27]. In the mouse mammary gland, activation of the transcription factor Stat3 by tyrosine phosphorylation has primarily been associated with inducing the transcription of genes involved in the initiation of cell death and tissue remodeling during mammary gland involution post lactation [38]. Previous analyses for phospho-Stat3 have shown that Stat3 is weakly activated in virgin mammary gland, turned off at the onset of pregnancy and reactivated for a short time at the end of pregnancy and the beginning of lactation. Stat3 signaling appears to remain silent for the duration of lactation until it is strongly reactivated at the beginning of involution [39]. In our studies we observed that phospho-Stat3 levels are significantly increased in Wwox KO mammary epithelium from virgin and pregnant mice (P18.5). Taking its role in involution into account we would expect to observe increased apoptosis due to the increased activated Stat3 but we saw no significant difference in cell death between WT and Wwox KO mammary glands (data not shown). In regards to tumorigenesis, despite having a pro-apoptotic function in mam- mary gland involution, Stat3 has been found to be constitutively active in breast cancers at low levels and these cancers appear to become addicted to Stat3 [39]. Further studies will be required to better understand the interplay between loss of WWOX function and Stat3 activation.

We also observed that the expression of several components of the canonical Wnt pathway were upregulated in Wwox KO mammary epithelium. Inhibition of WWOX has previously been associated with increased signaling through this pathway as shown by stabilization of the b-catenin protein as well as increased transcriptional activity in cell lines. It was shown that WWOX binds Dvl-2 and it was proposed that WWOX could play a role in sequestering such signal transducer in the cytoplasmic compartment [40]. Even though we observed an increase in the expression of b-catenin at gene expression level, we did not detect active bcatenin nuclear translocation nor did we observe an increase in the expression of known transcriptional target genes such as Cyclin D1, Myc or Fgf20 or increase in cell proliferation. Further experiments will be needed to clarify the effect of Wwox ablation on the canonical Wnt signaling pathway.

In summary, we report the first characterization of tissue specific targeted ablation of the putative tumor suppressor gene Wwox in adult mouse tissues. We demonstrated that conditional deletion of Wwox in the mouse mammary gland does not result in tumorigenicity. The fact that WWOX expression is lost in multiple neoplasias by a variety of mechanisms is undeniable and very well documented [4,5,7,41]. Thus, our data indicate that WWOX does not behave as a classical highly penetrant tumor suppressor gene and more likely the loss of WWOX expression is related to tumor progression. Experiments are in progress to determine whether Wwox ablation plays a role in mammary gland tumor progression. Generation of conditional Wwox knockout mice using BK5-Cre and MMTV-Cre mediated deletion Female Wwox flox/flox mice (129SV/C57Bl/6 background) were bred with male mice homozygous for the Cre recombinase gene under control of either the bovine keratin 5 (BK5) promoter or the MMTV promoter (Line D, [25]) to generate the Wwox +/DCre F1 generation. Generation of Wwox flox/flox mice is discussed elsewhere [19].  Mammary gland whole mount preparation, histology and morphometric analysis For whole mounts, the 4 th inguinal mammary glands were dissected, spread onto glass slides and fixed with formalin overnight and stained with carmine alum by standard procedures. Mammary gland whole mount slides were then scanned for image analysis and morphometry using the ScanScope CS system (Aperio Technologies, Inc., Vista, CA) and scans were analyzed for morphometric features such as number of branches per mm of ductal length.

Materials and Methods

Ethics statement

Immunohistochemistry

For immunohistochemistry, formalin-fixed paraffin sections from mammary glands were deparaffinized and tissue sections were treated with 10 mM Citrate buffer (pH 6.0; 15 min, microwave) for antigen unmasking and Biocare blocking reagent (Biocare) for blocking endogenous immunoglobulin in cases using primary antibodies. Staining was then performed using DAB (Dako) according to manufacturer's instructions. Primary antibodies used were rabbit anti-Wwox [14] (1:50 dilution), rabbit anti-pSTAT3 (Cell Signaling Technology, 1:50 dilution), rabbit anti-Estrogen Receptor alpha (Santa Cruz, 1:500 dilution), mouse anti-Progesterone Receptor (Abcam, 1:100 dilution) and rabbit anti-Ki67 (Bethyl, 1:250 dilution). Tissue sections were counterstained with hematoxylin. Quantification of cells that were positive for pSTAT3, ERa, PR or Ki67 was performed by scanning each stained section with the ScanScope system as described above. A thousand epithelial cells from random fields within the tissue section were counted to determine the percentage of cells that were positive for the respective staining.

Mammary gland transplantation experiments

Twelve week old virgin BK5-Cre Wwox KO mice served as mammary epithelium donors. Mammary tissues were minced into small pieces (,1 mm 3 ) and were used for transplantation into recipient mice. Recipient mice (n = 10) were 4 week old female SCID mice whose left and right 4 th mammary fat pads (MFPs) had been cleared as previously described [42]. The sites of the left MFPs were inoculated with mammary epithelial cells harvested from BK5-Cre Wwox-KO mice. Recipient mice were monitored for 10 months and transplanted MFPs were dissected. Analysis of MFPs was done by whole mount and histological examinations.

Microarray data processing, statistical and data mining analyses

Mammary gland epithelial organoids were obtained as previously described [43] from three WWOX-KO mice (BK5-cre+; WWOX flox/flox ) and from three WWOX-WT mice (BK5-cre -; WWOX flox/flox ). Total RNA was isolated and purified using the TRIzol/RNeasy Kit (Qiagen) from each mammary gland sample. RNA concentration and integrity were measured on an Agilent Bioanalyzer RNA 6000 Nanochip (Agilent Technologies). Five micrograms of total RNA per sample were used for cDNA synthesis, labeling and hybridization to whole genome Affymetrix GeneChip Mouse Genome 430 2.0 Arrays. Labeling and hybridization to Affymetrix GeneChips was carried out at the Genomics Core Facility at the MDACC. Briefly, we carried out QC and normalization procedures in R/Bioconductor [44] using the simpleaffy package [45].

To identify differentially expressed genes between the WWOX-WT vs. WWOX-KO mammary gland organoid expression profiles, we utilized the modified two-sample t-test using Limma package [46]. Raw datasets have been submitted to NCBI GEO database with accession number: GSE36665. Heatmap visualiza-tion of differentially expressed transcripts were done with the MultiExperiment Viewer software (MeV 4.8) [47]. For automated functional annotation and gene enrichment analysis, we used the Database for Annotation, Visualization and Integrated Discovery (DAVID) [48]. The DAVID resource calculates over-representation of specific biological themes/pathways with respect to the total number of genes assayed and annotated. REViGO resource was employed to summarize and visualize the enriched GO terms in a scatterplot graph based on the p-values obtained by DAVID. This allows the identification of biological themes/pathways within a specific list of differentially expressed genes.

RNA isolation and real time and semi-quantitative RT-PCR

Total RNA was isolated from mammary epithelial organoids from Wwox WT and KO female mice (n = 3 different mice in each group) using Trizol (Life Technologies) extraction and then cleaned up with Qiagen RNeasy Mini Kit; 300 ng of total RNA was used for reverse transcription using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). One uL of the 20 uL reverse transcription reaction was used for real-time PCR ran on the 7900HT real-time PCR system (Applied Biosystems). Taqman Gene Expression Assays (Applied Biosystems) for mouse Wwox (assay ID: Mm01247380_m1) and 18S rRNA (assay ID: Hs99999901_s1) were used in separate reactions to determine Wwox expression relative to 18S expression in each sample. Experiments were performed in triplicate. Wwox expression was normalized first to 18S expression within each sample and then to WT levels. For semi-quantitative RT-PCR 1 uL of cDNA reaction was added to a PCR reaction using the GoTaq Green Master Mix (Promega). Forward and reverse primers for mouse Wnt5a, Wwox and Gapdh were added to final concentration of 2 pg/uL and are as follows: Wnt5a F 59-TCCTTCGCCCAGGTTGTTAT-39, Wnt5a R 59-GCAGAGAGGCTGTGCACCTA-39, Wwox F 59-TGGAGCAAATTCGGGAATAG-39, Wwox R 59-ATTGCTTC-CACCTTGGCTTT-39, Gapdh F 59-CGTCCCGTAGA-CAAAATGGT-39, Gapdh R 59-TCAATGAAGGGGTCGTT-GAT-39. Reactions were run on a BioRad iCycler under standard conditions for 24, 28 and 32 cycles to ensure that reaction conditions were in linear range. PCR reaction products were electrophoresed on 2% agarose gel, stained with GelRed Nucleic Acid Stain (Phenix) and visualized under UV.

Western blot analysis

To monitor endogenous activation of the Stat pathway in mammary epithelium total protein extracts were prepared from the 4 th inguinal mammary epithelial organoids. Protein isolation was done using RIPA buffer (50 mM Tris buffer pH 7.5, 150 mM NaCl, 0.25% sodium deoxycholate, 10 mM EDTA, 1% NP40) with protease inhibitor cocktail (Roche, Mannheim, Germany) and the phosphatase inhibitors sodium vanadate (2 mM) and sodium fluoride (5 mM). For western blotting, 20 ug of total protein was separated on 12.5% SDS-PAGE and transferred to a PVDF membrane. Membranes were blocked and antibodies were diluted in 5% dry milk/TBST. Primary antibodies used were: rabbit anti-Wwox (1:2000) [7], rabbit anti-phospho-Stat3 (Santa Cruz, 1:1000) and mouse anti-Stat3 (Santa Cruz, 1:1000). HRP conjugated secondary antibodies were used followed by chemiluminescence autoradiography. Actin was used as the protein loading control and it was detected using monoclonal anti-actin antibody (Sigma, 1:1000) and HRP conjugated anti-mouse secondary antibody (1:5000). Immunodetection was performed using ECL Western Blotting Detection Reagents (GE Healthcare) as described by the manufacturer.

Supporting Information

Figure 1 .
1Conditional KO of Wwox via BK5-Cre and MMTV-Cre. (A-F) Immunostaining of formalin-fixed histological sections of mammary glands from each model of deletion reveals strong cytoplasmic Wwox staining in WT samples and significant loss of Wwox protein expression in KO animals. (A) 8 week virgin BK5 Wwox WT, (B) 8 week virgin BK5 Wwox KO, (C) P18.5 days BK5 Wwox WT, (D) P18.5 days BK5 Wwox KO, (E) 24 week virgin MMTV Wwox WT, (F)

Figure 2 .
2Wwox mRNA expression in BK5-Cre model of Wwox deletion. qRT-PCR was used to determine the mRNA expression of Wwox in mammary epithelial organoids from BK5 Wwox WT and KO animals. The box plots show dramatically decreased Wwox mRNA levels in epithelium from 8 week virgin (A) and P18.5 days (B) Wwox KO mammary epithelium when compared to their respective WT counterparts. Mammary epithelial organoids were isolated from 3 different WT and 3 different KO mice in each group. Samples from each mouse were run individually in triplicate to assess Wwox expression. Samples were normalized to 18S expression. doi:10.1371/journal.pone.0036618.g002

Figure 3 .
3Deletion of Wwox results in defective mammary branching and lobuloalveolar development. (A-B) Representative microphotographs obtained from whole mounts of 12 week virgin BK5 Wwox WT (A) and KO (B) mammary glands. The impaired branching phenotype was also dramatically evident in mammary gland from MMTV KO mice as illustrated in (C), extreme case of branching impairment in a 24 week virgin MMTV Wwox KO mammary gland. (D) The average number of branches/mm of ductal length was quantified for BK5-Cre (2); Wwox flox/flox (Cre (2) WT, n = 4), BK5-Cre (+); Wwox +/+ (Cre (+) WT, n = 4), and BK5-Cre (+); Wwox flox/flox (Wwox KO, n = 5). A small yet statistically insignificant (p = 0.06) decrease in branching can be seen in Cre(+) WT as compared to Cre(2) WT. The decreased branching found in Wwox KO mammary glands was significant when compared to either Cre (2) WT (p = 0.0002) or Cre (+) WT (p = 0.018). (E) Quantification of branches/mm ductal length in 24 week virgin MMTV Wwox WT controls (MMTV-Cre (2); Wwox flox/flox and MMTV-Cre (+); Wwox +/+ were grouped together, n = 4) and MMTV Wwox KO (MMTV-Cre(+); Wwox flox/flox , n = 4) mammary glands. Branching was statistically significantly decreased in Wwox KO when compared to WT controls (p = 0.017). Error bars indicate standard deviation. doi:10.1371/journal.pone.0036618.g003

Figure 4 .
4Wwox-KO BK5-Cre transgenic mice model gene expression profile study. (A) Heatmap of the differentially expressed genes between Wwox WT vs. Wwox KO mammary gland epithelial organoid samples (p,0.01; 2 Fold changes). Color scale at bottom of picture is used to represent expression level: low expression is represented by green, and high expression is represented by red. (B) Expression graph of the 913 deregulated probes (136 probes down-modulated and 777 up-modulated) among Wwox WT and Wwox KO mammary epithelial organoid samples. (C) Scatterplot graph showing the representative clusters, after redundancy reduction of the statistical significant GO terms (p,0.025) enriched in the deregulated gene list, in a two dimensional space related to GO terms' semantic similarities. Bubble color indicates the p-value of GO terms (expressed as Log10 p-value) and bubble size indicates the frequency of the GO term in the underlying GOA database (bubbles of more general terms are larger). doi:10.1371/journal.pone.0036618.g004

Figure 5 .
5Wnt5a expression increases in BK5 Wwox KO mammary epithelium. Semi-quantitative RT-PCR was performed on cDNA synthesized from RNA obtained from mammary epithelial organoids (3 different animals per group) as discussed in Materials and Methods. Lanes from left to right are: (lanes 1-3) 8 week virgin Wwox WT, (lanes 4-6) P18.5 Wwox WT, (lanes 7-9) 8 week virgin Wwox KO, and (lanes 10-12) P18.5 Wwox KO. Wwox expression is shown in the middle panel as further verification of Wwox ablation. Gapdh expression is shown as a normalization control. Each PCR reaction was performed at 24, 26, 28 and 32 cycles to ensure that reaction was in linear range. Results shown at 24 cycles. doi:10.1371/journal.pone.0036618.g005

Mice used in these studies were maintained in a clean, modifiedbarrier animal facility, fed regular commercial mouse diet (Harlan Lab, Indianapolis, IN) under controlled light (12L:12D) and temperature (68-74uF). All animal research was conducted in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International at the University of Texas, M.D. Anderson Cancer Center, Science Park, following international guidelines and all research was specifically approved by the University of Texas M. D. Anderson Cancer Center Institutional Animal Care and Use Committee, (Animal Welfare Assurance Number A3343-01).

BK5-Cre transgenic mice (129SV/C57Bl/6 background) were obtained from the laboratory of Dr. David Johnson (MDACC, Science Park) while the MMTV-Cre transgenic mice (129SV/ C57Bl/6 background) were obtained from Jackson Labs (stock #003553) Genotypes were determined by PCR using the oligonucleotide primers: Cre F: 59-GCCTGCATTACCGGTCGATG-CAACG-39 Cre R: 59-GTGGCAGATGGCGCGGCAACAC-CAT-39 Wwox-N1: 59-ATGGGCCGAAACTGGAGCTCAGA-A-39 Wwox-N2: 59-TCAGCAACTCACTCTGGCTTCAAC-39 Wwox-L: 59-GCATACATTATACGAAGTTATTCGAG-39.

Figure 6 .
6Wwox deletion correlates with increased phospho-Stat3 protein in mammary epithelium. (A-B) Immunostaining for phospho-Stat3 (pStat3) protein in histological sections from 12 week virgin BK5 Wwox WT (A) and Wwox KO (B) mammary glands. Strong nuclear staining can be seen in the majority of cells in Wwox KO sections. (C) Quantification of percentage of cells positive for pStat3 staining in Wwox WT (n = 3) and Wwox KO (n = 3) histological sections. A total of 1000 cells were counted from random fields within each sample. (D) Whole cell lysates from mammary epithelial organoids from P18.5 BK5 Wwox WT (n = 3; WT4, WT5, WT6) and Wwox KO (n = 3; KO4, KO5, KO6) mice, were probed pStat3 levels. Wwox protein levels are shown to verify Wwox knockdown. Total Stat3 levels are shown as a control and Actin protein levels are shown as a loading control. doi:10.1371/journal.pone.0036618.g006

Figure S1 Figure
S1S2 Cytokeratin 5 immunostaining in BK5 KO mammary gland (10 wk old virgin mouse). As can be observed, K5 staining is limited to the basal layer of every epithelial structure as in normal wild type mammary gland. No obvious abnormalities in epithelial differentiation were detected. (DOCX)

Table S1
S1List of 913 probes from Affymetrix gene expression profile differentially expressed between Wwox KO and Wwox WT mammary gland epithelium (p,0.01; 2 fold changes). (XLS)
PLoS ONE | www.plosone.org
May 2012 | Volume 7 | Issue 5 | e36618
AcknowledgmentsWe would like to thank Dr. John Ludes-Meyers for his contributions during the early stages of this project.Author Contributions
WWOX, a novel WW domain-containing protein mapping to human chromosome 16q23.3-24.1, a region frequently affected in breast cancer. A K Bednarek, K J Laflin, R L Daniel, Q Liao, K A Hawkins, Cancer Res. 60Bednarek AK, Laflin KJ, Daniel RL, Liao Q, Hawkins KA, et al. (2000) WWOX, a novel WW domain-containing protein mapping to human chromosome 16q23.3-24.1, a region frequently affected in breast cancer. Cancer Res 60: 2140-2145.

Common chromosomal fragile site FRA16D sequence: identification of the FOR gene spanning FRA16D and homozygous deletions and translocation breakpoints in cancer cells. K Ried, M Finnis, L Hobson, M Mangelsdorf, S Dayan, Hum Mol Genet. 9Ried K, Finnis M, Hobson L, Mangelsdorf M, Dayan S, et al. (2000) Common chromosomal fragile site FRA16D sequence: identification of the FOR gene spanning FRA16D and homozygous deletions and translocation breakpoints in cancer cells. Hum Mol Genet 9: 1651-1663.

The characterization of the common fragile site FRA16D and its involvement in multiple myeloma translocations. K A Krummel, L R Roberts, M Kawakami, T W Glover, D I Smith, Genomics. 69Krummel KA, Roberts LR, Kawakami M, Glover TW, Smith DI (2000) The characterization of the common fragile site FRA16D and its involvement in multiple myeloma translocations. Genomics 69: 37-46.

WWOX in biological control and tumorigenesis. R I Aqeilan, C M Croce, J Cell Physiol. 212Aqeilan RI, Croce CM (2007) WWOX in biological control and tumorigenesis. J Cell Physiol 212: 307-310.

WWOX, a chromosomal fragile site gene and its role in cancer. D Ramos, C M Aldaz, Adv Exp Med Biol. 587Ramos D, Aldaz CM (2006) WWOX, a chromosomal fragile site gene and its role in cancer. Adv Exp Med Biol 587: 149-159.

The common fragile site FRA16D and its associated gene WWOX are highly conserved in the mouse at Fra8E1. K A Krummel, S R Denison, E Calhoun, L A Phillips, D I Smith, Genes Chromosomes Cancer. 34Krummel KA, Denison SR, Calhoun E, Phillips LA, Smith DI (2002) The common fragile site FRA16D and its associated gene WWOX are highly conserved in the mouse at Fra8E1. Genes Chromosomes Cancer 34: 154-167.

WWOX, the common chromosomal fragile site, FRA16D, cancer gene. J H Ludes-Meyers, A K Bednarek, N C Popescu, M Bedford, C M Aldaz, Cytogenet Genome Res. 100Ludes-Meyers JH, Bednarek AK, Popescu NC, Bedford M, Aldaz CM (2003) WWOX, the common chromosomal fragile site, FRA16D, cancer gene. Cytogenet Genome Res 100: 101-110.

WWOX binds the specific proline-rich ligand PPXY: identification of candidate interacting proteins. J H Ludes-Meyers, H Kil, A K Bednarek, J Drake, M T Bedford, Oncogene. 23Ludes-Meyers JH, Kil H, Bednarek AK, Drake J, Bedford MT, et al. (2004) WWOX binds the specific proline-rich ligand PPXY: identification of candidate interacting proteins. Oncogene 23: 5049-5055.

PKA-mediated protein phosphorylation regulates ezrin-WWOX interaction. C Jin, L Ge, X Ding, Y Chen, H Zhu, Biochem Biophys Res Commun. 341Jin C, Ge L, Ding X, Chen Y, Zhu H, et al. (2006) PKA-mediated protein phosphorylation regulates ezrin-WWOX interaction. Biochem Biophys Res Commun 341: 784-791.

Nuclear localization of active HGF receptor Met in aggressive MDA-MB231 breast carcinoma cells. E Matteucci, P Bendinelli, M A Desiderio, Carcinogenesis. 30Matteucci E, Bendinelli P, Desiderio MA (2009) Nuclear localization of active HGF receptor Met in aggressive MDA-MB231 breast carcinoma cells. Carcinogenesis 30: 937-945.

Functional association between Wwox tumor suppressor protein and p73, a p53 homolog. R I Aqeilan, Y Pekarsky, J J Herrero, A Palamarchuk, J Letofsky, Proc Natl Acad Sci U S A. 101Aqeilan RI, Pekarsky Y, Herrero JJ, Palamarchuk A, Letofsky J, et al. (2004) Functional association between Wwox tumor suppressor protein and p73, a p53 homolog. Proc Natl Acad Sci U S A 101: 4401-4406.

WW domain-containing proteins, WWOX and YAP, compete for interaction with ErbB-4 and modulate its transcriptional function. R I Aqeilan, V Donati, A Palamarchuk, F Trapasso, M Kaou, Cancer Res. 65Aqeilan RI, Donati V, Palamarchuk A, Trapasso F, Kaou M, et al. (2005) WW domain-containing proteins, WWOX and YAP, compete for interaction with ErbB-4 and modulate its transcriptional function. Cancer Res 65: 6764-6772.

Physical and functional interactions between the Wwox tumor suppressor protein and the AP-2gamma transcription factor. R I Aqeilan, A Palamarchuk, R J Weigel, J J Herrero, Y Pekarsky, Cancer Res. 64Aqeilan RI, Palamarchuk A, Weigel RJ, Herrero JJ, Pekarsky Y, et al. (2004) Physical and functional interactions between the Wwox tumor suppressor protein and the AP-2gamma transcription factor. Cancer Res 64: 8256-8261.

WWOX, the FRA16D gene, behaves as a suppressor of tumor growth. A K Bednarek, C L Keck-Waggoner, R L Daniel, K J Laflin, P L Bergsagel, Cancer Res. 61Bednarek AK, Keck-Waggoner CL, Daniel RL, Laflin KJ, Bergsagel PL, et al. (2001) WWOX, the FRA16D gene, behaves as a suppressor of tumor growth. Cancer Res 61: 8068-8073.

WWOX gene restoration prevents lung cancer growth in vitro and in vivo. M Fabbri, D Iliopoulos, F Trapasso, R I Aqeilan, A Cimmino, Proc Natl Acad Sci U S A. 102Fabbri M, Iliopoulos D, Trapasso F, Aqeilan RI, Cimmino A, et al. (2005) WWOX gene restoration prevents lung cancer growth in vitro and in vivo. Proc Natl Acad Sci U S A 102: 15611-15616.

A role for the WWOX gene in prostate cancer. H R Qin, D Iliopoulos, S Semba, M Fabbri, T Druck, Cancer Res. 66Qin HR, Iliopoulos D, Semba S, Fabbri M, Druck T, et al. (2006) A role for the WWOX gene in prostate cancer. Cancer Res 66: 6477-6481.

WWOX gene expression abolishes ovarian cancer tumorigenicity in vivo and decreases attachment to fibronectin via integrin alpha3. C Gourley, A J Paige, K J Taylor, C Ward, B Kuske, Cancer Res. 69Gourley C, Paige AJ, Taylor KJ, Ward C, Kuske B, et al. (2009) WWOX gene expression abolishes ovarian cancer tumorigenicity in vivo and decreases attachment to fibronectin via integrin alpha3. Cancer Res 69: 4835-4842.

Targeted deletion of Wwox reveals a tumor suppressor function. R I Aqeilan, F Trapasso, S Hussain, S Costinean, D Marshall, Proc Natl Acad Sci U S A. 104Aqeilan RI, Trapasso F, Hussain S, Costinean S, Marshall D, et al. (2007) Targeted deletion of Wwox reveals a tumor suppressor function. Proc Natl Acad Sci U S A 104: 3949-3954.

Generation and characterization of mice carrying a conditional allele of the Wwox tumor suppressor gene. J H Ludes-Meyers, H Kil, J Parker-Thornburg, D F Kusewitt, M T Bedford, PLoS One. 47775Ludes-Meyers JH, Kil H, Parker-Thornburg J, Kusewitt DF, Bedford MT, et al. (2009) Generation and characterization of mice carrying a conditional allele of the Wwox tumor suppressor gene. PLoS One 4: e7775.

Frequent loss of WWOX expression in breast cancer: correlation with estrogen receptor status. M I Nunez, J Ludes-Meyers, M C Abba, H Kil, N W Abbey, Breast Cancer Res Treat. 89Nunez MI, Ludes-Meyers J, Abba MC, Kil H, Abbey NW, et al. (2005) Frequent loss of WWOX expression in breast cancer: correlation with estrogen receptor status. Breast Cancer Res Treat 89: 99-105.

Concordant loss of fragile gene expression early in breast cancer development. G Guler, A Uner, N Guler, S Y Han, D Iliopoulos, Pathol Int. 55Guler G, Uner A, Guler N, Han SY, Iliopoulos D, et al. (2005) Concordant loss of fragile gene expression early in breast cancer development. Pathol Int 55: 471-478.

Fragile histidine triad protein, WW domain-containing oxidoreductase protein Wwox, and activator protein 2gamma expression levels correlate with basal phenotype in breast cancer. G Guler, K Huebner, C Himmetoglu, R E Jimenez, S Costinean, Cancer. 115Guler G, Huebner K, Himmetoglu C, Jimenez RE, Costinean S, et al. (2009) Fragile histidine triad protein, WW domain-containing oxidoreductase protein Wwox, and activator protein 2gamma expression levels correlate with basal phenotype in breast cancer. Cancer 115: 899-908.

Association of Wwox with ErbB4 in breast cancer. R I Aqeilan, V Donati, E Gaudio, M S Nicoloso, M Sundvall, Cancer Res. 67Aqeilan RI, Donati V, Gaudio E, Nicoloso MS, Sundvall M, et al. (2007) Association of Wwox with ErbB4 in breast cancer. Cancer Res 67: 9330-9336.

A keratin K5Cre transgenic line appropriate for tissue-specific or generalized Cremediated recombination. A Ramirez, A Page, A Gandarillas, J Zanet, S Pibre, Genesis. 39Ramirez A, Page A, Gandarillas A, Zanet J, Pibre S, et al. (2004) A keratin K5Cre transgenic line appropriate for tissue-specific or generalized Cre- mediated recombination. Genesis 39: 52-57.

Spatial and temporal expression of the Cre gene under the control of the MMTV-LTR in different lines of transgenic mice. K U Wagner, K Mcallister, T Ward, B Davis, R Wiseman, Transgenic Res. 10Wagner KU, McAllister K, Ward T, Davis B, Wiseman R, et al. (2001) Spatial and temporal expression of the Cre gene under the control of the MMTV-LTR in different lines of transgenic mice. Transgenic Res 10: 545-553.

Differential regulation of the Wnt gene family during pregnancy and lactation suggests a role in postnatal development of the mammary gland. B J Gavin, A P Mcmahon, Mol Cell Biol. 12Gavin BJ, McMahon AP (1992) Differential regulation of the Wnt gene family during pregnancy and lactation suggests a role in postnatal development of the mammary gland. Mol Cell Biol 12: 2418-2423.

STAT3-induced WNT5A signaling loop in embryonic stem cells, adult normal tissues, chronic persistent inflammation, rheumatoid arthritis and cancer (Review). M Katoh, Int J Mol Med. 19Katoh M (2007) STAT3-induced WNT5A signaling loop in embryonic stem cells, adult normal tissues, chronic persistent inflammation, rheumatoid arthritis and cancer (Review). Int J Mol Med 19: 273-278.

High basal levels of functional toll-like receptor 3 (TLR3) and noncanonical Wnt5a are expressed in papillary thyroid cancer and are coordinately decreased by phenylmethimazole together with cell proliferation and migration. K D Mccall, N Harii, C J Lewis, R Malgor, W B Kim, Endocrinology. 148McCall KD, Harii N, Lewis CJ, Malgor R, Kim WB, et al. (2007) High basal levels of functional toll-like receptor 3 (TLR3) and noncanonical Wnt5a are expressed in papillary thyroid cancer and are coordinately decreased by phenylmethimazole together with cell proliferation and migration. Endocrinol- ogy 148: 4226-4237.

Frequent attenuation of the WWOX tumor suppressor in osteosarcoma is associated with increased tumorigenicity and aberrant RUNX2 expression. K C Kurek, Del Mare, S Salah, Z Abdeen, S Sadiq, H , Cancer Res. 70Kurek KC, Del Mare S, Salah Z, Abdeen S, Sadiq H, et al. (2010) Frequent attenuation of the WWOX tumor suppressor in osteosarcoma is associated with increased tumorigenicity and aberrant RUNX2 expression. Cancer Res 70: 5577-5586.

Wwox inactivation enhances mammary tumorigenesis. S K Abdeen, Z Salah, B Maly, Y Smith, R Tufail, Oncogene. 30Abdeen SK, Salah Z, Maly B, Smith Y, Tufail R, et al. (2011) Wwox inactivation enhances mammary tumorigenesis. Oncogene 30: 3900-3906.

Inactivation of the Wwox gene accelerates forestomach tumor progression in vivo. R I Aqeilan, J P Hagan, H A Aqeilan, F Pichiorri, L Y Fong, Cancer Res. 67Aqeilan RI, Hagan JP, Aqeilan HA, Pichiorri F, Fong LY, et al. (2007) Inactivation of the Wwox gene accelerates forestomach tumor progression in vivo. Cancer Res 67: 5606-5610.

Purified Wnt5a protein activates or inhibits betacatenin-TCF signaling depending on receptor context. A J Mikels, R Nusse, PLoS Biol. 4115Mikels AJ, Nusse R (2006) Purified Wnt5a protein activates or inhibits beta- catenin-TCF signaling depending on receptor context. PLoS Biol 4: e115.

Autonomous regulation of osteosarcoma cell invasiveness by Wnt5a/Ror2 signaling. M Enomoto, S Hayakawa, S Itsukushima, D Y Ren, M Matsuo, Oncogene. 28Enomoto M, Hayakawa S, Itsukushima S, Ren DY, Matsuo M, et al. (2009) Autonomous regulation of osteosarcoma cell invasiveness by Wnt5a/Ror2 signaling. Oncogene 28: 3197-3208.

Wnt5a signaling is involved in the aggressiveness of prostate cancer and expression of metalloproteinase. H Yamamoto, N Oue, A Sato, Y Hasegawa, A Matsubara, Oncogene. 29Yamamoto H, Oue N, Sato A, Hasegawa Y, Matsubara A, et al. (2010) Wnt5a signaling is involved in the aggressiveness of prostate cancer and expression of metalloproteinase. Oncogene 29: 2036-2046.

Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma. A T Weeraratna, Y Jiang, G Hostetter, K Rosenblatt, P Duray, Cancer Cell. 1Weeraratna AT, Jiang Y, Hostetter G, Rosenblatt K, Duray P, et al. (2002) Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma. Cancer Cell 1: 279-288.

Wnt5a is required for proper mammary gland development and TGF-beta-mediated inhibition of ductal growth. K Roarty, R Serra, Development. 134Roarty K, Serra R (2007) Wnt5a is required for proper mammary gland development and TGF-beta-mediated inhibition of ductal growth. Development 134: 3929-3939.

Transcriptional mechanisms of WNT5A based on NF-kappaB, Hedgehog, TGFbeta, and Notch signaling cascades. M Katoh, Int J Mol Med. 23Katoh M (2009) Transcriptional mechanisms of WNT5A based on NF-kappaB, Hedgehog, TGFbeta, and Notch signaling cascades. Int J Mol Med 23: 763-769.

Suppression of epithelial apoptosis and delayed mammary gland involution in mice with a conditional knockout of Stat3. R S Chapman, P C Lourenco, E Tonner, D J Flint, S Selbert, Genes Dev. 13Chapman RS, Lourenco PC, Tonner E, Flint DJ, Selbert S, et al. (1999) Suppression of epithelial apoptosis and delayed mammary gland involution in mice with a conditional knockout of Stat3. Genes Dev 13: 2604-2616.

Stat3 and the inflammation/acute phase response in involution and breast cancer. S Pensa, C J Watson, V Poli, J Mammary Gland Biol Neoplasia. 14Pensa S, Watson CJ, Poli V (2009) Stat3 and the inflammation/acute phase response in involution and breast cancer. J Mammary Gland Biol Neoplasia 14: 121-129.

Inhibition of the Wnt/beta-catenin pathway by the WWOX tumor suppressor protein. N Bouteille, K Driouch, P E Hage, S Sin, E Formstecher, Oncogene. 28Bouteille N, Driouch K, Hage PE, Sin S, Formstecher E, et al. (2009) Inhibition of the Wnt/beta-catenin pathway by the WWOX tumor suppressor protein. Oncogene 28: 2569-2580.

WWOX, the tumour suppressor gene affected in multiple cancers. U Lewandowska, M Zelazowski, K Seta, M Byczewska, E Pluciennik, J Physiol Pharmacol. 60SupplLewandowska U, Zelazowski M, Seta K, Byczewska M, Pluciennik E, et al. (2009) WWOX, the tumour suppressor gene affected in multiple cancers. J Physiol Pharmacol 60 Suppl 1: 47-56.

Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H mice. K B Deome, L J FaulkinJr, H A Bern, P B Blair, Cancer Res. 19Deome KB, Faulkin LJ Jr., Bern HA, Blair PB (1959) Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H mice. Cancer Res 19: 515-520.

Serial analysis of gene expression in normal p53 null mammary epithelium. C M Aldaz, Y Hu, R Daniel, S Gaddis, F Kittrell, Oncogene. 21Aldaz CM, Hu Y, Daniel R, Gaddis S, Kittrell F, et al. (2002) Serial analysis of gene expression in normal p53 null mammary epithelium. Oncogene 21: 6366-6376.

Bioconductor: open software development for computational biology and bioinformatics. R C Gentleman, V J Carey, D M Bates, B Bolstad, M Dettling, Genome Biol. 580Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, et al. (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5: R80.

Simpleaffy: a BioConductor package for Affymetrix Quality Control and data analysis. C L Wilson, C J Miller, Bioinformatics. 21Wilson CL, Miller CJ (2005) Simpleaffy: a BioConductor package for Affymetrix Quality Control and data analysis. Bioinformatics 21: 3683-3685.

limma: Linear Models for Microarray Data. G K Smyth, Bioinformatics and Computational Biology Solutions Using R and Bioconductor. Springer VerlagSmyth GK (2005) limma: Linear Models for Microarray Data. Bioinformatics and Computational Biology Solutions Using R and Bioconductor. New York: Springer Verlag. pp 397-420.

TM4: a free, opensource system for microarray data management and analysis. A I Saeed, V Sharov, J White, J Li, W Liang, Biotechniques. 34Saeed AI, Sharov V, White J, Li J, Liang W, et al. (2003) TM4: a free, open- source system for microarray data management and analysis. Biotechniques 34: 374-378.

Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. W Huang Da, B T Sherman, R A Lempicki, Nat Protoc. 4Huang da W, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4: 44-57.


Prevalence of Dental Caries in Kosovar Adult Population

Blerim Kamberi firstname.lastname@example.com
Department of Dental Pathology and Endodontics
University Dentistry Clinical Center of Kosovo
10000Prishtina, Kosovo

Ferit Koçani
Department of Dental Pathology and Endodontics
University Dentistry Clinical Center of Kosovo
10000Prishtina, Kosovo

Agim Begzati
Department of Pediatric and Preventive Dentistry
University Dentistry Clinical Center of Kosovo
10000Prishtina, Kosovo

Jeta Kelmendi
Department of Orthodontics
University Dentistry Clinical Center of Kosovo
10000Prishtina, Kosovo

Donika Ilijazi
Private Dental Clinic
10000Prishtina, Kosovo

Nora Berisha
Department of Orthodontics
University Dentistry Clinical Center of Kosovo
10000Prishtina, Kosovo

Lumnije Kqiku
Division of Preventive and Operative Dentistry
Endodontics, Pedodontics and Minimally Invasive Dentistry
Department of Dentistry and Maxillofacial Surgery
University of Graz
8010GrazAustria

Prevalence of Dental Caries in Kosovar Adult Population
10.1155/2016/4290291Received 8 March 2016; Revised 18 May 2016; Accepted 26 June 2016 Academic Editor: Manal AwadResearch Article Correspondence should be addressed to Blerim Kamberi;
Objectives. The aim of this study was to assess the prevalence of dental caries in the Kosovar adult population. Materials and Methods. A cross-sectional study in Kosovo was conducted examining 9387 patients, aged 18 upwards, between January 2010 and December 2011. Clinical evaluation was done using WHO criteria for evaluation of dental health status and data collection. Results. The prevalence of caries for the whole study was 72.80%. The mean DMFT index was 9.61 (±5.12) in the 18-34-year age group, 11.6 (±6.42) in the 35-44-year age group, 13.68 (±8.12) among the 45-64-year age group, 17.98 (±9.81) in the 65-74-year age group, and 23.19 (±9.41) in the age group of 75+ years, respectively. A significant difference of mean DMFT and its each component was observed between the ages ( < 0.001). Conclusion. This study comes out with the significant levels of dental caries among young Kosovar population (18-34 years old).Materials and Methods2.1. Subjects. This was a cross-sectional study comprising 9387 patients from different regions of Kosovo aged 18-75+.

Introduction

Dental caries continues to be a major health concern for populations worldwide. Damage caused by caries leads to a decrease in the quality of life of the affected individuals and high economic costs for both individuals and society, with disparities related to well-known issues of socioeconomics, immigration, lack of preventive efforts, and dietary changes [1]. Despite the overall decline in caries prevalence in developed countries [2], caries continues to be an important disease in most developing countries. Studies have shown that caries remains a major problem in the adult population of both developing and industrialized countries [3][4][5].

Evaluation of the oral health status in the adult age group is important because it presents important information for planning services in dental care and also generates data on the outcomes of dental care provided to the population during their lifetime [6].

In Kosovo, no studies have been conducted which include examination by dentists to determine the prevalence of caries or tooth loss among adults. Most epidemiological studies have focused mainly on children rather than adults [7]. Therefore, strategies that are needed to treat affected people remain unknown. Data collected in this study may be useful to build a more complete profile of the oral health status of the adult population of Kosovo and will supplement the analysis of global trends of the disease. Therefore, the objective of this study was to assess the prevalence and severity of dental caries in the Kosovar adult population in relation to their sex, age, and place of residence (urban or rural). The University Dentistry Clinical Center of Kosovo is the only secondary and tertiary care center for the whole territory of Kosovo. Within each year, a large number of populations use the free services at the center, hence providing a heterogeneous population sample. It was due to the latter fact that the authors decided to use population samples from this center's dental records for this study.

Data

Collection. Data were collected based on age, sex, and residence (rural or urban). The exclusion criteria were patients under the age of 18 and those who were missing any of the required information.

For calibration, six examiners were trained by an experienced dentist prior to the clinical examinations. The interexaminer agreement determined by Cohen's Kappa values was 0.7-0.9.

In the WHO criteria used for clinical evaluation of health status and data collection, only the evaluation of hard tissue of the teeth was included [8]. Patients were examined under an artificial light, using a dental mirror and a dental probe. All present teeth were taken into consideration during the clinical examination. Diagnosis of dental caries was made according to the criteria recommended by the WHO [8] (i.e., when a lesion in a pit or fissure or on a smooth tooth surface had an unmistakable cavity, undermined enamel, or a detectably softened floor or wall). Caries severity was measured by DMFT index, which records the number of DT (decayed teeth), MT (missing teeth), and FT (filled teeth).

Data Analysis.

Clinical data were first entered into modified WHO Oral Health Status [8] paper forms, then entered into Excel, and subsequently exported to Statistical Package for Social Sciences (SPSS 17.0 for Windows, 13.0 Program Package) for statistical analysis. The prevalence proportion rates, mean values, and standard deviations were calculated for the purpose of analysis. Statistical analysis was performed applying descriptive statistics and the independent sample Chi-square test, Fisher Exact test between different groups, and one-way ANOVA tests to compare the means of DMFT, DT, MT, and FT to the other variables. The level of statistical significance was set at = 0.05. Figure 1 shows characteristics of the subjects by sex and group. The number of female participants was 5337 (59.5%), while the number of males was 4050 (40.5%). Table 1 shows the subjects according to sex and residence. A total of 70.9% of subjects lived in the city while 29.1% lived in the countryside. According to sex, 73.3% of females and 67.6% of males lived in the city.

Results

The highest percentage of participants diagnosed with caries was aged 18-34 years (4152 participants, 44.2%), while the lowest percentage was for participants aged 75 years and above (2.9%). The other age groups had similar participation rates: 18.3% for patients aged 35-44 years, 17.2% for patients aged 45-64 years, and 17.3% for patients aged 65-74 years. Our data show significant differences between sex and residence and between sex and age ( < 0.001). Table 2 shows the presence of caries according to sex, age, and residence.

According to the sex and caries distribution, 3903 patients (57.1%) with caries were female, while 2929 (42.9%) patients with caries were male.

Patients diagnosed with caries, 69.6%, lived in the city, compared with 30.4% who lived in the countryside. The age group with the highest prevalence of caries was the 18-34year age group (54%), and the age group with the lowest prevalence was the age group of 75+ years (0.8%). Using the Chi-square test, we compared caries prevalence according to sex and found no significant difference for = 0.354. However, when we compared caries prevalence according to age with caries prevalence according to residence, there was a significant difference in both comparisons for < 0.001. Table 3 shows the DMFT and caries prevalence according to age and sex. The prevalence of caries for the whole study was 72.80%, and it decreased with age: 18-34 years (88.80%), 34-44 years (77.90%), 45-64 years (65.50%), 65-74 years (42.40%), and 75+ years (20.90%). There was no significant difference between males and females.

The mean DMFT (SD) value for the study group was 12.52 (±7.91). The DMFT value increased with age from 9.61 (±5.12) for 18-34 years to 11.6 (±6.42) for 35-44 years, to 13.68 (±8.12) for 45-64 years, to 17.98 (±9.87) for 65-74 years, and to 23.19 (±9.41) for 75+ years.

The analysis of the DMFT components showed that the MT score was the major component.

These variables were also tested with ANOVA, and, as shown in the table for the DMFT index, there is no significant difference between the means according to sex ( = 0.613). There was also no significant difference between the means according to sex for the DT and MT components, but there was a significant difference between the mean values according to sex for the FT component ( < 0.001).

Our results show significant differences between the means according to age in the DMFT index and all of its components with < 0.001.    Table 4 shows that caries prevalence was slightly higher for patients living in rural areas (75.9%) than for patients living in urban areas (71.5%).

The highest mean value and SD were for the MT component with a value of 7.31 ± 8.83, followed by the DT component with a mean value of 3.15 ± 3.31, and the FT component with the lowest mean value of 2.05 ± 2.86.

Comparing the mean values of the DMFT index and its components according to the patients' residence, we found no significant difference for the MT component with = 0.617.

Patients from an urban residence had a higher DMFT index compared with those from a rural residence with a significant difference for < 0.05.

Comparing the averages of the DT and FT components according to residence using one-way ANOVA test, we found that the average DT component was higher among the rural population (3.44 ± 3.45) than among the urban population (3.03 ± 3.24) with no significant difference for < 0.001, although the average FT component was higher among the urban population (3.01 ± 2.26) than among the rural 4 International Journal of Dentistry

Discussion

Most studies about dental caries in Kosovo have focused on children under 12 years of age, and the state of dental caries in adults has remained unknown. A previous study assessing the prevalence and analyzing caries risk factors, in the Kosovar population between 2002 and 2005, had included measurements of early childhood caries, DEFT, and DMFT [7]. This is the first study that has investigated the epidemiology of dental caries among adults in Kosovo.

In agreement with most studies [9][10][11], it was found that the prevalence of caries was slightly higher in females than in males (Table 2). However, the present study did not observe any significant sex differences in caries prevalence that was confirmed in earlier findings [12,13].

According to WHO recommendations, oral health surveillance of oral health conditions in adults should include the 35-44-year age group and the 65-74-year age group [8]. In our study, in addition to these two age groups, we included the age groups of 18-34 years, 45-64 years, and over 75 years (Figure 1). The prevalence of dental caries in the 18-34-year age group in the present study was found to be the highest. This outcome is thought to be a result of poor socioeconomic development, lack of preventive services in health insurance, and inadequate knowledge about the importance of oral health as a consequence of the absence of oral health promotion programs in adults.

The caries prevalence for the 35-44-year age group has been reported in many studies [14][15][16]. In the present study, the prevalence of dental caries in this age group was found to be 77.90%, which is lower than in some previous studies [14,15,17] [5]. Our results showed that as age increased, the prevalence of caries decreased, which is in contrast with other research findings showing that as age increases so does the prevalence of caries [18]. This holds true since with age the prevalence of missing teeth increases compared to the prevalence of decayed and filled teeth.

Differences in the prevalence rates may be due to different study criteria. Observed differences in the caries prevalence of the adult population suggest that it may be possible to develop and implement oral health policies taking into account geographical and socioeconomic differences in populations [19].

Differences in the prevalence of caries between urban and rural areas are narrowing as the socioeconomic development in rural areas increases. Therefore, the prevalence of caries in some rural areas was even higher than in urban areas [20], which could be a result of lower level of oral health education and socioeconomic status. However, our study shows no significant difference in caries prevalence between urban and rural subjects (Table 4), which is in accordance with a study in the United States [21].

The mean DMFT for the study sample was 12.52 and increased among the ages (Table 3) and is consistent with the results of other studies [22,23].

By the latest data, the mean DMFT value observed among 18-34-year age group was 9.61, compared to a value of 4.37 in an 18-25-year age group [24] in a neighbouring region, Italy, and 12.76 in a 20-34-year age group [19] in Hungary; our score shows moderate condition among this population, which means a relative "average position" for Kosovo.

In the 35-44-year age group, the mean DMFT value was 11.6, which was a better score than in many countries; DMFT scores for the same age group were 16.1 in Germany [25], 15.4 in Belgium [26], 14.7 in Austria [27], 15.4 in Hungary [19], and 14.4 in Norway [28]. However, the mean DMFT score in our study for individuals aged 65-74 years (17.98) was higher than in many other countries [19,25,27].

Comparisons of the mean DMFT findings with those of other studies should be undertaken with caution, since there is great variation in the findings in the literature. This discrepancy may be due to differences in geographic location, population culture, diagnostic criteria, and sampling procedures.

The value of F/DMFT observed in our study was 2.47 for the 35-44-year age group and only 0.77 for the 65-74year age group. These values are significantly lower than those in certain European countries such as Spain, Denmark, Austria, and France, where patients have a higher proportion of fillings than in Kosovo [17,27,29,30]. A potential reason for this might be that people in Kosovo tend to seek dental care when they experience severe dental pain rather than for routine dental checkups; hence, by the time they seek dental care, the caries is at an advanced stage. In most cases, the tooth has to be extracted because it is beyond restoration or requires complex and expensive endodontic treatment. The high percentage of untreated teeth in individuals aged 65-74 years may be due to difficulties in accessing dental services and a lack of interest in dental health or due to other health conditions.

It was also noted that the DT component decreased with age while the MT component increased with age. Reasons for the high prevalence of decayed teeth in adults aged 18-34 years, together with the low prevalence of filled teeth in comparison with other European countries, are mainly a consequence of the health insurance system in Kosovo.

Fillings and endodontic treatment are free for about 40% of the Kosovar population covered by the private health insurance system, which still does not include free preventive measures and guidelines for adults. In summary, the high prevalence of decayed, missing, and filled teeth in the adult population of Kosovo also reflects the lack of preventive services in the state health insurance system.

There is a high DT component and there is a very low FT component of the DMFT, mainly because of the lack of restorative dental services or to some extent low utilization of this service.

As a consequence of the absence of the data, we were not able to compare our results with neighboring states.

Conclusion

The present study demonstrated significant levels of dental caries among young Kosovar population (18-34 years old) where carious and missing teeth due to caries are predominant.

It is understandable that Kosovo, as a new state with major economic and social problems and with a developing health care department, has a significant number of priorities to balance with the dental-oral health care of the population. Therefore, based on the WHO recommendations, to improve the general oral health of the population, the focus should be on prevention, starting with health education, promotion of oral health, and application of preventive measures in the Kosovar population.

Further studies of dental health that cover more regions of Kosovo will help to identify public dental health problems, an essential step in improving the general health status of the citizens of this country [31].

Figure 1 :
1Patients participation according to sex and age group.

Table 1 :
1Patients participation according to sex and residence.Sex
F
M
T o t a l
value
%
%
%
Residence
Rural
1425
26.7
1311
32.4
2736
29.1
< 0.001
Urban
3912
73.3
2739
67.6
6651
70.9

Table 2 :
2Caries presence according to gender, age, and residence.Caries
No
Yes
Total
value
%
%
%
Gender
Female
1434
56.1
3903
57.1
5337
56.9
= 0.354
Male
1123
43.9
2927
42.9
4050
43.1
Age group
18-34
467
18.3
3685
54
4152
44.2

< 0.001

35-44
380
14.9
1337
19.6
1717
18.3
45-64
558
21.8
1061
15.5
1619
17.2
65-74
936
36.6
690
10.1
1626
17.3
75+
216
8.4
57
0.8
273
2.9
Residence
Rural
659
25.8
2077
30.4
2736
29.1
< 0.001
Urban
1898
74.2
4753
69.6
6651
70.9

Table 3 :
3DMFT, D, M, and F and caries prevalence according to sex and age group.DMFT
D
M
F
Caries prevalence
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Sex
Female
12.56
7.682
3.25
3.339
7.1
8.538
2.21
2.962
73.10%
Male
12.47
8.206
3.03
3.268
7.6
9.214
1.84
2.725
72.30%
Total
12.52
7.912
3.15
3.31
7.31
8.839
2.05
2.868
72.80%
= 0.613
= 0.002
= 0.007
< 0.001
Age group
18-34
9.61
5.123
4.48
3.528
2.47
3.161
2.66
3.129
88.80%
35-44
11.6
6.425
3.18
3.087
5.95
5.878
2.47
3.067
77.90%
45-64
13.68
8.125
2.2
2.607
9.85
8.546
1.63
2.479
65.50%
65-74
17.98
9.876
1.14
1.923
16.06
10.8
0.77
1.688
42.40%
75+
23.19
9.419
0.53
1.453
22.35
10.095
0.32
0.972
20.90%
Total
12.52
7.912
3.15
3.31
7.31
8.839
2.05
2.868
72.80%
< 0.001
< 0.001
< 0.001
< 0.001

Table 4 :
4DMFT, D, M, and F and caries prevalence according to residence.DMFT
D
M
F
Caries prevalence
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Residence
Rural
12.23
7.862
3.44
3.459
7.24
8.631
1.54
2.411
75.90%
Urban
12.64
7.93
3.03
3.24
7.34
8.924
2.26
3.011
71.50%
Total
12.52
7.912
3.15
3.31
7.31
8.839
2.05
2.868
72.80%
= 0.022
< 0.001
= 0.617
< 0.001

population (1.54 ± 2.41) with a highly significant difference
for < 0.001.

but higher than in the studies conducted by Doifode et al. (48.6%) [16] and Namal et al. (58.2%)
AcknowledgmentsThis study was supported by and obtained the data from the University Dentistry Clinical Center of Kosovo conducted by the Ministry of Health.Ethical ApprovalThe protocols and the human data that we used in this study were approved by the Ethical Board of the University Dentistry Clinical Center of Kosovo.Competing InterestsThe authors would like to declare that there are no competing interests related to this paper.
The global increase in dental caries. A pending public health crisis. R A Bagramian, F Garcia-Godoy, A R Volpe, American Journal of Dentistry. 221R. A. Bagramian, F. Garcia-Godoy, and A. R. Volpe, "The global increase in dental caries. A pending public health crisis, " American Journal of Dentistry, vol. 22, no. 1, pp. 3-8, 2009.

The World Oral Health Report 2003: continuous improvement of oral health in the 21st century-the approach of the WHO Global Oral Health Programme. P E Petersen, Community Dentistry and Oral Epidemiology. 311P. E. Petersen, "The World Oral Health Report 2003: continuous improvement of oral health in the 21st century-the approach of the WHO Global Oral Health Programme, " Community Den- tistry and Oral Epidemiology, vol. 31, no. 1, pp. 3-24, 2003.

Ten-year incidence of dental caries in adult and elderly Chinese. W.-M Luan, V Baelum, O Fejerskov, X Chen, Caries Research. 343W.-M. Luan, V. Baelum, O. Fejerskov, and X. Chen, "Ten-year incidence of dental caries in adult and elderly Chinese, " Caries Research, vol. 34, no. 3, pp. 205-213, 2000.

Factors associated with oral health: a multivariate analysis of results from the 1998 adult dental health survey. E Treasure, M Kelly, N Nuttall, J Nunn, G Bradnock, D White, British Dental Journal. 1902E. Treasure, M. Kelly, N. Nuttall, J. Nunn, G. Bradnock, and D. White, "Factors associated with oral health: a multivariate analysis of results from the 1998 adult dental health survey, " British Dental Journal, vol. 190, no. 2, pp. 60-68, 2001.

Dental health status and risk factors for dental caries in adults in Istanbul, Turkey. N Namal, G Can, S Vehid, S Koksal, A Kaypmaz, Eastern Mediterranean Health Journal. 141N. Namal, G. Can, S. Vehid, S. Koksal, and A. Kaypmaz, "Dental health status and risk factors for dental caries in adults in Istanbul, Turkey, " Eastern Mediterranean Health Journal, vol. 14, no. 1, pp. 110-118, 2008.

Dental Caries: The Disease and Its Clinical Management. O Fejerskov, E A M Kidd, Blackwell MunksgaardOxford, UKO. Fejerskov and E. A. M. Kidd, Dental Caries: The Disease and Its Clinical Management, Blackwell Munksgaard, Oxford, UK, 2008.

Dental health evaluation of children in Kosovo. A Begzati, K Meqa, D Siegenthaler, M Berisha, W Mautsch, European Journal of Dentistry. 51A. Begzati, K. Meqa, D. Siegenthaler, M. Berisha, and W. Mautsch, "Dental health evaluation of children in Kosovo, " European Journal of Dentistry, vol. 5, no. 1, pp. 32-39, 2011.

World Health Organization, Oral Health Surveys: Basic Methods, World Health Organization. Geneva, SwitzerlandWorld Health Organization, Oral Health Surveys: Basic Meth- ods, World Health Organization, Geneva, Switzerland, 1997.

Dental caries' experience, prevalence and severity in Mexican adolescents and young adults. J O García-Cortés, C E Medina-Solís, J P Loyola-Rodriguez, Revista de Salud Publica. 111J. O. García-Cortés, C. E. Medina-Solís, J. P. Loyola-Rodriguez et al., "Dental caries' experience, prevalence and severity in Mexican adolescents and young adults, " Revista de Salud Pub- lica, vol. 11, no. 1, pp. 82-91, 2009.

Prevalence and severity of dental caries in adolescents aged 12 and 15 living in communities with various fluoride concentrations. A P Pontigo-Loyola, C E Medina-Solis, S A Borges-Yañez, N Patiño-Marín, A Islas-Márquez, G Maupome, Journal of Public Health Dentistry. 671A. P. Pontigo-Loyola, C. E. Medina-Solis, S. A. Borges-Yañez, N. Patiño-Marín, A. Islas-Márquez, and G. Maupome, "Prevalence and severity of dental caries in adolescents aged 12 and 15 living in communities with various fluoride concentrations, " Journal of Public Health Dentistry, vol. 67, no. 1, pp. 8-13, 2007.

Socioeconomic inequalities in oral healt: dental caries in 6 to 12 year-old children. C E Medina-Solís, G Maupomé, B Pelcastre-Villafuerte, L Avila-Burgos, A A Vallejos-Sánchez, A J Casanova-Rosado, Revista de Investigacion Clinica. 584C. E. Medina-Solís, G. Maupomé, B. Pelcastre-Villafuerte, L. Avila-Burgos, A. A. Vallejos-Sánchez, and A. J. Casanova- Rosado, "Socioeconomic inequalities in oral healt: dental caries in 6 to 12 year-old children, " Revista de Investigacion Clinica, vol. 58, no. 4, pp. 296-304, 2006.

Impact of socio-demographic variables, oral hygiene practices, oral habits and diet on dental caries experience of Indian elderly: a community-based study. N Shah, K R Sundaram, Gerodontology. 211N. Shah and K. R. Sundaram, "Impact of socio-demographic variables, oral hygiene practices, oral habits and diet on dental caries experience of Indian elderly: a community-based study, " Gerodontology, vol. 21, no. 1, pp. 43-50, 2004.

Epidemiology of dental caries among adults in a rural area in India. A M Maru, S Narendran, Journal of Contemporary Dental Practice. 133A. M. Maru and S. Narendran, "Epidemiology of dental caries among adults in a rural area in India, " Journal of Contemporary Dental Practice, vol. 13, no. 3, pp. 382-388, 2012.

Risk indicators of coronal and root caries in Greek middle aged adults and senior citizens. E Mamai-Homata, V Topitsoglou, C Oulis, V Margaritis, A Polychronopoulou, BMC Public Health. 121484E. Mamai-Homata, V. Topitsoglou, C. Oulis, V. Margaritis, and A. Polychronopoulou, "Risk indicators of coronal and root caries in Greek middle aged adults and senior citizens, " BMC Public Health, vol. 12, no. 1, article 484, 2012.

Caries prevalence in an adult population: results of the Study of Health in Pomerania, Germany (SHIP). C Splieth, C Schwahn, O Bernhardt, Oral Health & Preventive Dentistry. 12C. Splieth, C. Schwahn, O. Bernhardt et al., "Caries prevalence in an adult population: results of the Study of Health in Pomera- nia, Germany (SHIP), " Oral Health & Preventive Dentistry, vol. 1, no. 2, pp. 149-155, 2003.

Assessment of oral health status and its association with some epidemiological factors in population of Nagpur, India. V V Doifode, N N Ambadekar, A G Lanewar, Indian Journal of Medical Sciences. 547International Journal of DentistryV. V. Doifode, N. N. Ambadekar, and A. G. Lanewar, "Assess- ment of oral health status and its association with some epidemiological factors in population of Nagpur, India, " Indian Journal of Medical Sciences, vol. 54, no. 7, pp. 261-269, 2000. International Journal of Dentistry

Oral health survey of the adult population of the Valencia region (Spain). M.-V Eustaquio, J.-M Montiel, J.-M Almerich, Patologia Oral. 153Cirugia BucalM.-V. Eustaquio, J.-M. Montiel, and J.-M. Almerich, "Oral health survey of the adult population of the Valencia region (Spain), " Medicina Oral, Patologia Oral, Cirugia Bucal, vol. 15, no. 3, pp. e538-e544, 2010.

Oral health of Singapore adults. T Loh, J Chan, C N Low, Singapore Dental Journal. 211T. Loh, J. Chan, and C. N. Low, "Oral health of Singapore adults, " Singapore Dental Journal, vol. 21, no. 1, pp. 6-10, 1996.

Caries prevalence and tooth loss in Hungarian adult population: results of a national survey. M Madléna, P Hermann, M Jáhn, P Fejérdy, BMC Public Health. 8M. Madléna, P. Hermann, M. Jáhn, and P. Fejérdy, "Caries prevalence and tooth loss in Hungarian adult population: results of a national survey, " BMC Public Health, vol. 8, article 364, 2008.

Socioeconomic factors that affect international caries levels. D E Diehnelt, H A Kiyak, Community Dentistry and Oral Epidemiology. 293D. E. Diehnelt and H. A. Kiyak, "Socioeconomic factors that affect international caries levels, " Community Dentistry and Oral Epidemiology, vol. 29, no. 3, pp. 226-233, 2001.

Oral health status of older rural adults in the United States. C M Vargas, J A Yellowitz, K L Hayes, The Journal of the American Dental Association. 1344C. M. Vargas, J. A. Yellowitz, and K. L. Hayes, "Oral health status of older rural adults in the United States, " The Journal of the American Dental Association, vol. 134, no. 4, pp. 479-486, 2003.

Dental caries in Quebec adults aged 35 to 44 years. J.-M Brodeur, M Payette, M Benigeri, P F Gagnon, M Olivier, D Chabot, Journal of the Canadian Dental Association. 667J.-M. Brodeur, M. Payette, M. Benigeri, P. F. Gagnon, M. Olivier, and D. Chabot, "Dental caries in Quebec adults aged 35 to 44 years, " Journal of the Canadian Dental Association, vol. 66, no. 7, pp. 374-379, 2000.

Changes in caries experience among Australian public dental patients between 1995/96 and 2001/02. D S Brennan, A J Spencer, Australian and New Zealand Journal of Public Health. 286D. S. Brennan and A. J. Spencer, "Changes in caries experience among Australian public dental patients between 1995/96 and 2001/02, " Australian and New Zealand Journal of Public Health, vol. 28, no. 6, pp. 542-548, 2004.

The influence of educational level and oral hygiene behaviours on DMFT index and CPITN index in an adult Italian population: an epidemiological study. M Vano, S Gennai, D Karapetsa, International Journal of Dental Hygiene. 132M. Vano, S. Gennai, D. Karapetsa et al., "The influence of edu- cational level and oral hygiene behaviours on DMFT index and CPITN index in an adult Italian population: an epidemiological study, " International Journal of Dental Hygiene, vol. 13, no. 2, pp. 151-157, 2015.

Caries in adolescents, adults and seniors in Germany. U Schiffner, E Reich, Caries Research. 33286U. Schiffner and E. Reich, "Caries in adolescents, adults and seniors in Germany, " Caries Research, vol. 33, p. 286, 1999.

Application of the CPITN in a group of 35-65 Years-old Belgian employees. Comparison with different parameters. P Bercy, J Meurisse, M Lambert, R Tonglet, Journal Dental Research. 77831P. Bercy, J. Meurisse, M. Lambert, and R. Tonglet, "Application of the CPITN in a group of 35-65 Years-old Belgian employ- ees. Comparison with different parameters, " Journal Dental Research, vol. 77, article 831, 1998.

Caries prevalence in a 35-44 and 65-74 year-old Austrian population. P Städtler, A Bodenwinkler, G Sax, Caries Research. 362007P. Städtler, A. Bodenwinkler, and G. Sax, "Caries prevalence in a 35-44 and 65-74 year-old Austrian population, " Caries Research, vol. 36, p. 2007, 2002.

Tannhelseutviklingen i den voksne befolkningen i Nord-Trøndelag fra. D Holst, A Schuller, K Dahl, Norske Tannlegeforenings Tidende. 11D. Holst, A. Schuller, and K. Dahl, "Tannhelseutviklingen i den voksne befolkningen i Nord-Trøndelag fra 1973 til 2006, " Norske Tannlegeforenings Tidende, vol. 11, pp. 804-811, 2007.

Oral health status of adults in Denmark. U Krustrup, P Petersen, Community Dental Health. 19202U. Krustrup and P. Petersen, "Oral health status of adults in Denmark, " Community Dental Health, vol. 19, p. 202, 2002.

Oral health in 35-44 year old adults in France. P Hescot, D Bourgeois, J Doury, International Dental Journal. 472P. Hescot, D. Bourgeois, and J. Doury, "Oral health in 35-44 year old adults in France, " International Dental Journal, vol. 47, no. 2, pp. 94-99, 1997.

Prevalence of apical periodontitis and endodontic treatment in a Kosovar adult population. B Kamberi, V Hoxha, M Stavileci, E Dragusha, A Kuçi, L Kqiku, BMC Oral Health. 11132B. Kamberi, V. Hoxha, M. Stavileci, E. Dragusha, A. Kuçi, and L. Kqiku, "Prevalence of apical periodontitis and endodontic treat- ment in a Kosovar adult population, " BMC Oral Health, vol. 11, no. 1, article 32, 2011.