the multistage model of mouse skin carcinogenesis is a useful system in which biochemical events unique to initiation , promotion , or progression steps of carcinogenesis can be studied and related to cancer formation . 12-o - tetradecanoylphorbol-13-acetate ( tpa ) , a component of croton oil , is a potent mouse skin tumor promoter [ 1 , 2 ] . a major breakthrough in understanding the mechanism of tpa tumor promotion has been the identification of protein kinase c ( pkc ) , as its major intracellular receptor . pkc forms part of the signal transduction system involving the turnover of inositol phospholipids and is activated by dag , which is produced as a consequence of this turnover . pkc represents a family of phospholipid - dependent serine / threonine kinases [ 36 ] . pkc is among the six pkc isoforms (  ,  ,  ,  ,  , and  ) expressed in both mouse and human skin . we have reported that epidermal pkc levels dictate the susceptibility of pkc transgenic ( tg ) mice to the development of squamous cell carcinomas ( scc ) elicited either by repeated exposures to ultraviolet radiation ( uvr )   or initiation with 7 , 12-dimethylbenz[a]anthracene ( dmba ) and tumor promotion with 12-o - tetradecanoylphorbol-13-acetate ( tpa ) . histologically , scc in tg mice , like human scc , is poorly differentiated and metastatic . the papilloma - independent carcinomas which develop in pkc transgenic mice arise from the hair follicle and have increased metastatic potential . the difference in metastatic potential and the different origin of malignancy when compared to wt provided support for the hypothesis that papilloma - independent carcinomas in pkc tg mice were pathologically distinct from wt mouse carcinomas . although the papilloma - independent carcinomas appeared to originate from the hair follicle , it was possible that the origin of the tumor was not within the hair follicle . however , this did not appear to be the case because we observed neoplastic cells arising only from the hair follicle and not the epidermis . by harvesting pkc tg and wt mice after 8 weeks of dmba + tpa or dmba + acetone treatments , we identified possible premalignant areas in pkc transgenic mice as early as 8 weeks after dmba + tpa treatment . the premalignant lesions originated within the hair follicle . the metastatic potential of a transformed keratinocyte appeared to inversely correlate with the differentiation potential of that keratinocyte in the limited number of tumors studied to date . this conclusion was based on the location of invasion and pathological categorization of pkc tg mouse carcinomas compared with wt mouse carcinomas . evidence suggests that these cells appear to be the stem or progenitor cells for both the hair follicle and epidermis and , therefore , would be in a less - differentiated state than other epidermal cells the carcinomas of pkc tg mice that led to metastases were also less differentiated than carcinomas from wt mice . evidence suggests that malignant cells invading from the hair follicle were less differentiated and had a higher metastatic potential than cells that invaded from the epidermis . pkc , when activated either via direct binding to tpa or indirectly by uvr treatment , mediates two potential signals leading to inhibition of apoptosis [ 11 , 12 ] and induction of cell proliferation . epidermal stem cells in the mouse hair follicle are known to be the precursor cells for scc in the mouse skin [ 1317 ] . evidence suggests that epithelial stem cells reside in the bulge region [ 18 , 19 ] . stem cells , unlike transit amplifying cells , are slowly cycling and thus seem probable target cells . moreover morris et al .   demonstrated that label retaining cells ( lrcs ) have another property characteristic of potential initiated cells : they could retain carcinogen - dna adducts . the contribution of follicular and interfollicular stem cells to the induction of skin papillomas and carcinomas was also determined . however , only follicular stem cells were linked to the development of carcinomas . as a prelude to determine the scc lineage from hscs in pkc tg mice , we compared the responses of pkc tg and their wt littermates to uvr treatment . we examined the effects on proliferation , turnover , and gene expression profile of hscs . in this communication , we present for the first time that ( 1 ) uvr exposures increased the number of double positive hscs in tg mice , ( 2 ) the percent of double positive hscs retaining brdu label in the wt mice was 7-fold more than the tg mice , indicating that the double positive cells in the tg mice cycle at a faster rate , ( 3 ) the keratinocytes from pkc tg mice have higher proliferating potential compared to their wt littermates , and ( 4 ) a comparison of gene expression profile of facs - sorted hscs showed an increase expression of pes1 , rad21 , tfdp1 , and cks1b genes in tg mice compared to their wt littermates . brdu was purchased from sigma aldrich ( st . louis , mo , usa ) . brdu antibody was purchased from santa cruz biotechnologies ( santa cruz , ca , usa ) . antibodies used for facs such as 6-integrin pe - conjugated , cd34 fitc - conjugated antibodies , apc brdu labeling kit , and propidium iodide were purchased from bd biosciences ( san jose , ca , usa ) . brdu antibody conjugated with alexa fluor 647 was procured from biosciences ( frederick , md , usa ) . pcr gene array focused to cell cycle was purchased from sa biosciences ( frederick , md , usa ) . keratinocytes were harvested as described elsewhere . in each experiment an equal size of skin is excised from the wt and tg mice . keratinocytes were incubated for 1  hr in the dark at 4c with pe - conjugated rat anti - human 6-integrin antibody at 10  l per 10 cells and fitc - conjugated rat antimouse cd34 antibody at 2  g per 10 cells ( pe-6-integrin and fitc - cd34 antibodies ; bd biosciences ) . keratinocyte preparations were sorted based on 6-integrin+ and cd34 status using a facs aria cell sorter ( bd biosciences ) . cells were stained with pe - conjugated anti-6-integrin and fitc - conjugated anti - cd34 antibodies for flow cytometry . a 488  nm laser was used to detect fitc with a 530/30 filter and a 532  nm laser for pe with a 575/25 filter . the nozzle size was 130  nm and the pressure used was 14  p.s.i . the live cell population gate was estimated using forward and side scatter positioning and confirmed with 7aad staining . keratinocytes were harvested from the dorsal skin of 7 - 8 weeks old pkc overexpressing mice ( tg224 and tg215 ) and their wt littermates . the skin hairs were clipped and the skin pieces trypsinized for 2  hrs at 32c . three thousand cells per dish were seeded onto irradiated 3t3 cells in 60  mm dishes and cultured for 2 weeks in high calcium medium . for feeder layer , 3t3 cells were cultured in emem medium with 10%  fbs and 1% penicillin - streptomycin and irradiated in cesium gamma irradiator at 5000  rad . irradiated 3t3 cells seeded 10 cells / dish to the 60  mm dishes a day before seeding keratinocytes . the clonal culture was grown in william 's e media with 10%  fbs and supplements . for counting and measurement of colonies , after fixation , the cultures were stained with 0.5% rhodamine b for 30  min to visualize colonies . wt and pkc tg 224 and 215 mice lines ( fvb background ) described elsewhere [ 9 , 10 ] were housed in groups of two to three in plastic bottom cages in light- , humidity- , and temperature - controlled rooms ; food and water were available ad libitum . the animals were kept in a normal rhythm of 12  h light and 12  h dark periods . the uvr source was kodacel - filtered fs-40 sun lamps ( approximately 60%  uvb and 40%  uva ) . mice were used for experimentation starting at 5 to 6 weeks of age . for 24  hr , 48  hr , and 72 hr time points , the mice were treated for 10  min ( 2  kj / m ) and skin was harvested for keratinocyte isolation after uv exposure . however , for multiple or chronic uvr exposures , mice were exposed to uvr ( 2  kj / m ) three times weekly ( monday , wednesday , and friday ) or a total of 8 times .   to identify the label retaining cells ( lrcs ) , newborn mice ( 3 days old ) were injected subcutaneously with brdu ( 50  mg / kg body weight ) twice daily for 3 days . mice were sacrificed 3 to 8 weeks after brdu injection ( 3 , 4 , 5 , 6 , and 8 weeks ) . freshly harvested keratinocytes were incubated with pe - conjugated anti-6-integrin and fitc - conjugated anti - cd34 antibodies , fixed and stained using the apc brdu flow kit , following the manufacturer 's instructions ( bd biosciences , san jose , ca , usa ) . to prepare brdu positive control samples for facs , 5-week - old female mice were injected brdu ( 50  mg / kg body weight ) intraperitoneally for two days ( 2 times each ) . after two days , the spleen and thymus were harvested for brdu positive cells and used as a positive control . for cell cycle analysis , freshly harvested keratinocytes were isolated from mouse dorsal skin and stained with pe - conjugated 6-integrin and fitc - conjugated anti - cd34 antibodies . after surface staining , the cells were fixed and then stained overnight with dapi for cell cycle analysis . flow cytometric analysis based on 6-integrin , cd34 , and brdu was performed on a lsrii benchtop flow cytometer ( bd biosciences ) . a multilength ultraviolet laser along with a 450/50 bandpass filter was used to detect dapi . dapi was used for dna staining for live / dead determinations along with cell cycle . for the detection of apc - conjugated anti - brdu antibody , a 640  nm laser and a 660/20 filter was used . the phenotyping assays were acquired on a bd facscalibur ( bd biosciences ) , and brdu assays were acquired on an lsr ii ( bd biosciences ) benchtop flow cytometer . both instruments were calibrated daily by the university of wisconsin carbone cancer center flow cytometry laboratory staff using the manufacturer 's cytometer settings and tracking calibration software . data were analyzed using flowjo software version 9.4.3 ( treestar , ashland , or , usa ) . dead cells were excluded using 7-aminoactinomycin d ( 7aad ) staining on facs calibur assays or invitrogen live / dead fixable violet ( flvd ) staining for brdu assays acquired on the bd lsr ii . data demonstrate frequency of cells in a parent population of live intact cells for 6-integrin and cd34 expression and of 6-integrin+/cd34+/live intact cells for brdu incorporation . the frequency of cd34+/6-integrin+ stem cells represents the percent of cd34+/6-integrin+/7aad- cells (  cells  determined by the absolute number of cd34+/6-integrin+ cells in individual samples was calculated by multiplying frequency of cd34+/6-integrin+ stem cells by the total number of trypan - blue excluding cells in the single cell keratinocyte preparation . the data represent absolute number of cd34+/6-integrin+ stem cells from the equal size of dorsal skin from wt and tg mice used in the study . similarly , for calculation for the absolute count of cd34+/6-integrin+/brdu+ cells , the total frequency of cd34+/6-integrin+/brdu+/fldv - cells in total flvd- was multiplied by the total counts of trypan - blue excluding cells in the single cell keratinocyte preparation . to identify the lrcs , newborn mice ( 3 days old ) were injected subcutaneously with brdu ( 50  mg / kg body weight ) twice daily for 3 days . mice were sacrificed at 3 , 4 , 5 , 6 , and 8 weeks after brdu injection . mouse skin was then excised promptly after euthanasia and immediately placed in 10% neutral - buffered formalin for fixation and then embedded in paraffin . four to five  m sections were cut for immunohistochemistry of brdu and k15 . for immunofluorescence study extra paraffin was removed using three xylene gradient washes followed by alcohol gradient ( 95% , 90% , 70% , 50% , and 30% ) for 10  min each . the antigen retrieval was done using antigen unmasking solution as per the protocol ( vector laboratories ) . the blocking process was done in normal goat and normal horse serum for 1  hr at room temperature ( rt ) . after blocking , primary antibodies ( keratin-15 monoclonal from neomarkers , ca , dilution 1  :  30 ) and brdu ( santa cruz , dilution 1  :  50 ) were incubated to tissue section on the slides for overnight . tissue sections were incubated with their secondary antibodies for 1  hr at rt such as alexa - fluor 488-donkey antimouse igg ( h + l ) and alexa - fluor 594-donley antirat igg ( h + l ) for k15 and brdu from invitrogen , respectively . after incubation with secondary antibody slides were washed three times with 1xpbs , mounted with dapi , and observed under the fluorescent microscope ( vectra ) . tg mice and their wt littermates were exposed to uv once ( 2  kj / m ) , and 24  hrs after uv treatment mice were sacrificed and the dorsal skin removed for keratinocyte isolation . the cells were sorted into 5  ml tubes containing 0.5  ml of heat inactivated , chelated , fetal bovine serum . the collected cells were then spun down and placed into 300  ml of rnaprotect cell reagent ( qiagen ; valencia , ca , usa ) . rna was isolated from double positive hscs using sa biosciences rt qpcr grade isolation kit ( sa biosciences , frederick , md , usa ) . 250  ng rna was used for first - strand cdna synthesis with the sa biosciences rt firststrand kit . the resulting cdna was used in the sa biosciences cell cycle gene array according to the manufacturer 's instructions . the real - time expression primers of pkc in the study were selected from origene website and further checked in ncbi primer blast for their primer - specific details such as proper target binding and amplification product ( http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?link_loc=blasthome ) . the samples were treated with dnase to remove the dna contamination using qiagen rnase - free dnase set ( qiagen ) . for cdna synthesis superscript first - strand synthesis kit ( invitrogen ) was used as per the manufacturer ' protocol .   briefly a total of 50  l reaction mixture consisted of 25  l ; 2x faststart universal sybr green master ( rox ) mix , 30.0  m ( 1  l ) forward and reverse primers , and pcr grade water , and 50100  ng of cdna ( 510  l ) was used . final volume of the reaction was adjusted with rnase - free water provided with the kit . a cycle to threshold ( ct ) value was assigned automatically at the beginning of the logarithmic phase of real - time pcr . finally , differences in ct value of control ( mouse gapdh ) and stem cell samples were used to determine the relative gene expression or fold changes of the pkc. to determine the basal levels of double positive hscs ( cd34+/6-integrin+ ) in untreated wt , tg224 , and tg215 mice , freshly harvested keratinocytes were labeled with cd34 and 6-integrin antibodies and analyzed by flow cytometry for their total frequency . we observed higher frequency and absolute count of total hscs in tg215 mice compared to wt and tg224 mice ( figures 1(a ) and 1(b ) ) . clonogenicity is an intrinsic property of adult stem cells . in this experiment ( figures 1(c ) and 1(d ) ) , an equal number of isolated keratinocytes from wt , tg224 , and tg215 mice were seeded onto the irradiated 3t3 ( fibroblast ) cells and left for two weeks . we observed increased colony formation in keratinocytes isolated from tg224 and tg215 mice compared with their wt littermates indicating more proliferative potential ( figures 1(c ) and 1(d ) ) . notably , the colonies greater than 2  mm and total numbers of colonies were higher in tg215 mice compared to their wt littermates ( figure 1(d ) ) . the cell surface markers cd34 and 6-integrin mark mouse hair follicle bulge cells , which have attributes of stem cells , including quiescence and multipotency . we determined the effects of uv treatment on total number of hscs . in this experiment , pkc tg and wt mice were exposed to a single or chronic uv doses ( 1.8  kj / m , monday , wednesday , and friday ) . at the indicated times after last uv exposure , mice were sacrificed and the number of putative hscs was determined by flow cytometric analysis . the total frequency as well as absolute count of double positive hscs were increased at 48 and 72  hr in tg224 after - uv exposure ( figures 2(a ) and 2(b ) ) and 24 , 48 , and 72  hr in tg215 ( figures 2(c ) and 2(d ) ) compared to their wt littermates . as shown in figures 3(a ) and 3(b ) , chronic uv exposures also showed an increase in total double positive hscs in both tg224 and tg215 mice compared to their wt littermates . to determine the proliferation rates of bulge region stem cells in wt and tg mice , a 5-bromo-2-deoxyuridine labeling ( brdu ) experiment was performed . in this experiment , three - day - old neonatal mice were injected with 50  mg / kg of brdu in pbs twice daily for three days . at 3 , 4 , 5 , 6 , and 8 weeks after brdu injections , mice were sacrificed and the dorsal skin excised for keratinocyte isolation . immunohistochemistry results revealed that , at 3-week time point , brdu labeling was prominent in bulge region of hair follicle , interfollicular epidermis , and sebaceous glands of tg 224 , 215 , and their wt littermates ( figures 4(a)4(c ) ) . however , at later time points ( 6 and 8 weeks ) , brdu labeling was decreased in tg mice compared to wt mice and localized to bulge region only ( figures 4(d)4(h ) ) . it is interesting to note that even after 3 weeks , some cells of interfollicular epidermis are able to retain the brdu label ( figures 4(a)4(c ) ) . moreover , dual immunofluorescence staining of brdu and k15 indicates the colocalization of brdu with k15 expressing cells in the stem cell - specific compartment , that is , bulge ( figures 4(i)4(k ) ) . we further evaluated the cell cycle pattern in sorted double positive hscs in tg 215 and their wt littermates at 8 weeks after - brdu injection . the percent of brdu - labeled cells was different in wt and tg215 mice . in the wt mice , the percent of double positive cells maintaining brdu label was 28.4  0.6% compared to 4.0  0.06% for the tg , an approximately 7-fold decrease ( figure 5(a ) ) . we further determined the turnover of hscs in wt and tg 224 mice . in this experiment , we analyzed the brdu retaining double positive hscs in isolated keratinocytes from wt and tg mice . brdu retaining cells were analyzed at 4 , 5 , and 8 weeks after - brdu injections . the frequency of brdu retaining double positive hscs was at 4 weeks ( wt = 0.246% , tg = 0.0807% ) , 5 weeks ( wt = 0.0364% , tg = 0.00337% ) , and 8 weeks ( wt = 0.167% , tg = 0.008% ) . there was also a decrease in total frequency as well as absolute count of brdu retaining double positive hscs in tg224 mice compared to wt littermates ( figures 5(b ) and 5(c ) ) . we first analyzed the percent distribution of cd34 + , cd34+/6-integrin+ ( double positive hscs ) , 6-integrin+ , and cd34/6-integrin- ( double negative ) in tg224 , tg215 , and their wt littermates . the 6-integrin+ cells were 70.9% , 62.7% , and 54.8% in wt , tg224 , and tg215 mice , respectively . the cd34 + cells were 2.8% , 4.0% , and 3.1% in wt , tg224 , and tg215 mice , respectively ( figure 6(a ) ) . we analyzed the pkc mrna expression levels in facs - sorted keratinocytes ( figure 6(b ) ) . the higher expression of pkc was recorded in double positive hscs of tg215 mice compared to wt and tg224 ( figure 6(b ) ) .   a possibility explored that an increased turnover of hscs in tg mice may be the result of changes in specific genes . in this experiment ( table 1 ) , the effect of uv on cell cycle - related genes in double positive hscs of tg and wt was determined using a focused cell cycle pcr array . a comparison of gene expression profiles of double positive hscs is shown in table 1 . a 1.7- to 3.2-fold increase in the expression of pes1 , rad21 , tfdp1 , and cks1b genes was observed in tg215 mice compared to their wt littermates . however , downregulation of ccnf , cdkn1a ( p21 ) , pkd-1 , and taf10 was observed in tg215 mice as compared to wt littermates . chronic exposure of sun 's uv radiation is linked to the development of human scc , a metastatic nonmelanoma skin cancer . we found using a novel pkc tg mouse model that the pkc levels in epidermis dictate the susceptibility of transgenic mice to the induction of scc by uv . pkc tg mice , when exposed to uv ( 2  kj / m thrice weekly ) , elicited 3-fold increased scc multiplicity and decreased tumor latency by 12 weeks . pkc overexpression in mice suppressed uv - induced sunburn ( apoptotic ) cell formation and enhanced both uv - induced hyperplasia and levels of specific cytokines ( tumor necrosis factor  ( tnf ) , granulocyte colony - stimulating factor ( g - csf ) , granulocyte macrophage colony - stimulating factor , and interleukin six ( il-6 ) ) , implying inhibition of apoptosis and promotion of preneoplastic cell survival [ 8 , 23 ] . additionally , pkc may impart sensitivity to uvr carcinogenesis via its association with stat3 , a transcriptional factor that is constitutively activated in both mouse and human scc . we now present that pkc-mediated susceptibility to uv carcinogenesis may involve stimulation of putative hscs proliferation possibly mediated by pkc and other specific genes linked to the cell cycle regulation . the epidermis undergoes a continual renewal throughout life , and the process is facilitated by various stem cell localized in both interfollicular epidermis and other specialized stem cell niches such as bulge . the skin stem cells present in different compartment of hair follicles respond differently to various signals mediated by their microenvironment . interestingly , multiple skin stem cell populations exist in the epidermis and play an important role during the process of controlled proliferation and differentiation ( reviewed in ) . the major stem cell population of hair follicle includes interfollicular label retaining cells ( lrcs ) , double positive hscs ( cd34+/6-integrin+ ) , mts24 + cells , blimp1 , nestin , lgr5 + , and lgr6 + cells ( reviewed in ) . however , the bulge region of hair follicle is considered as the major niche for keratinocyte stem cells [ 27 , 28 ] . particularly , the cd34+/6-integrin+ cells are slow cycling and colocalize with lrcs and confined to bulge region of hair follicles . in terms of their colony forming ability ( clonogenicity ) , cd34 + cells make larger colonies compared to cd34 cells [ 16 , 29 ] . keratinocytes isolated from pkc overexpressing tg have higher frequency of double positive hscs ( cd34+/6-integrin+ ) and clonogenicity than their wt littermates ( figure 1 ) . uv treatment resulted in a modest increase in total double positive hscs in both tg224 and tg215 mice compared to their wt littermates ( figure 2 ) . uv treatment in tg mice as compared to wt mice leads to constitutive activation of stat3 , increased stat3-dna binding , and increased expression of tnf and g - csf . results from genetic experiments indicate that both stat3 and tnf are linked to uv - induced development of scc . it yet remains to be proven that pkc downstream components stat3 and tnf directly affect the proliferation of putative hscs . we observed less brdu retaining double positive hscs cells in tg mice compared to wt ( figure 3 ) . evidence indicates that at least two types of cell population exist : the slow cycling designated as stem cells and rapidly cycling cells as transit amplifying cells [ 3032 ] . rapid turnover of double positive hscs cells in tg mice may be the effect of overexpression of pkc in tg mice and its associated cytokines such as tnf and g - csf . additionally , the level or percentage of brdu retention in the stem cell populations is not consistently uniform . brdu retention is influenced by mice age , time of labeling , and site of labeling . this may be the possible explanation that the amount of brdu retained varies in different repeat experiments ( figure 3 ) . however , brdu retaining double positive hscs cells were consistently less in tg mice compared to wt . an analysis of focused cell cycle cdna array revealed up- and downregulation of specific genes . the genes found to be overexpressed in double positive hscs in tg215 were pescadillo , tfdp-1 , rad21 , nfatc1 , cks1b , ak1 , and itgb1 . interestingly , nfatc1 gene is found to be overexpressed in many cancers , and its loss is linked with constant hair cycling and no quiescence . nfatc1 is also responsible for the balance between the quiescence and proliferation stage of skin stem cells . other overexpressed genes , pescadillo , rad21 , cks1b , ak1 , and itgb1 , are also linked with the process of carcinogenesis ( table 1 ) . in summary , we present for the first time an association of pkc with hscs , the scc precursors . pkc overexpression in mice increased the clonogenicity of isolated keratinocytes , a property commonly ascribed to stem cells . both single and chronic uv - treatments resulted in an increase in the frequency of double positive hscs in pkc tg mice as compared to their wt littermates . in tg mice , hscs cycle at a faster rate as compared to wild - type mice . a comparison of gene expression profiles of facs - sorted double positive keratinocytes isolated from uv - treated wt and tg mice indicated increased expression of pes1 , rad21 , tfdp1 , and cks1b genes in tg mice linked to cell transformation , invasion , and metastasis of cancer cells . it is believed that the skin stem cells are the major targets of carcinogen [ 15 , 41 ] . however , the identification and the precise location of cancer initiating cells in cutaneous scc is not clear . furthermore , the role of nonstem cell can not be overlooked during the process of carcinogenesis . it has been observed that the differentiated , nondividing epidermal cells with activated mapk kinase 1 and inflammatory infiltrate can initiate benign tumor formation . interestingly , the differentiated keratinocytes can reenter into active cell cycling , dedifferentiating , and acquiring the stemness . in future it will be interesting to study the link of double positive hscs and other skin stem cell populations , along with other inflammatory signals in uv - induced squamous cell carcinoma .
to find clues about the mechanism by which kinase c epsilon ( pkc ) may impart susceptibility to ultraviolet radiation ( uvr)-induced development of cutaneous squamous cell carcinomas ( scc ) , we compared pkc transgenic ( tg ) mice and their wild - type ( wt ) littermates for ( 1 ) the effects of uvr exposures on percent of putative hair follicle stem cells ( hscs ) and ( 2 ) hscs proliferation . 
 the percent of double hscs ( cd34 + and 6-integrin or cd34+/cd49f+ ) in the isolated keratinocytes were determined by flow cytometric analysis . both single and chronic uvr treatments ( 1.8  kj / m2 ) resulted in an increase in the frequency of double positive hscs in pkc tg mice as compared to their wt littermates . to determine the rate of proliferation of bulge region stem cells , a 5-bromo-2-deoxyuridine labeling ( brdu ) experiment was performed . in the wt mice , the percent of double positive hscs retaining brdu label was 28.4  0.6% compared to 4.0  0.06% for the tg mice , an approximately 7-fold decrease . 
 a comparison of gene expression profiles of facs sorted double positive hscs showed increased expression of pes1 , rad21 , tfdp1 and cks1b genes in tg mice compared to wt mice . 
 also , pkc over expression in mice increased the clonogenicity of isolated keratinocytes , a property commonly ascribed to stem cells .