Recent evidence suggests that an initial barrier to the emergence of tumours is a DNA damage resp... more Recent evidence suggests that an initial barrier to the emergence of tumours is a DNA damage response that evokes a counter-response which arrests the growth of, or eliminates, damaged cells. Early precursor lesions express markers of an activated DNA damage response in several types of tumour, with a diminishing response in more advanced cancers. An important marker of DNA damage is ATM which becomes phosphorylated (pATM) upon activation. We have investigated pATM expression patterns in cultured keratinocytes, skin explants and a spectrum of pre-malignant to malignant keratinocyte skin lesions by immunohistochemistry. We found that pATM was mainly localised to the Golgi apparatus, which contrasts with its nuclear localisation in other tissues. Upon UV irradiation there is transient formation of pATM in nuclear foci, consistent with recruitment to the sites of DNA damage. By immunohistochemistry we show pATM expression in precancerous keratinocyte lesions is greater and predominantly nuclear when compared to the invasive lesions where pATM is weaker and predominantly cytoplasmic. Our results are consistent with the hypothesis that the DNA damage response acts as a barrier to cutaneous tumour formation, but also suggests that ATM expression in skin is different compared to other tissues. This may be a consequence of the constant exposure of skin to UVR, and has implications for skin carcinogenesis.
Skin Cancer after Organ Transplantation, Jan 1, 2009
Human papillomaviruses (HPV) are small double-stranded DNA viruses that predominantly form hyperp... more Human papillomaviruses (HPV) are small double-stranded DNA viruses that predominantly form hyperproliferative lesions in mucosal and cutaneous epithelial tissues and are strictly epitheliotropic. More than 120 different types have been characterised based on DNA ...
Recent studies strongly support a role of human papillomavirus type 8 (HPV8) in non-melanoma skin... more Recent studies strongly support a role of human papillomavirus type 8 (HPV8) in non-melanoma skin cancer development. In this study, a quantitative two-dimensional (2D) differential gene expression (DiGE) gel approach combined with mass spectrometry has been used to identify proteins that are abundantly deregulated in primary human epidermal keratinocytes expressing HPV8 sequences. Twenty six protein spots showed significant changes in the level of expression between keratinocytes expressing E7 or the complete early region (CER) of HPV8 compared to extracts from cells lacking HPV8 gene expression. No differences between HPV8 E7 alone and HPV8 CER expressing cells were observed. The 26 protein spots that were differentially expressed corresponded to 20 different proteins, of which 14 actin-associated proteins were downregulated except for calponin-2, which was the only actin-binding protein that was overexpressed. Besides changes in actin modulating proteins, an upregulation of cytokeratins (CK) 5, 6 and 14 was also noted. This study suggests that the actin and keratin cytoskeleton modulating proteins are targets for HPV8.
Human papillomaviruses (HPVs) are small DNA viruses, which specifically infect keratinocytes at d... more Human papillomaviruses (HPVs) are small DNA viruses, which specifically infect keratinocytes at different body sites. The association between cutaneous squamous cell carcinoma (SCC) formation, ultraviolet (UV) irradiation and infection with a high-risk subset of cutaneous HPVs has been postulated although the underlying molecular mechanisms by which HPV may play a role in SCC development are not yet fully elucidated. Expression of the viral E6 oncoprotein has been shown to interfere with DNA damage responses and inhibit UV-induced apoptosis, suggesting HPV can contribute to early stages in tumorigenesis. However, cutaneous SCCs, in contrast to HPV-associated anogenital cancers do not harbor HPV DNA in every tumor cell. Here, we show that expression of E6 from the prototypic skin cancer-associated HPV type 5 induced the secretion of factors that were able to inhibit UV-induced apoptosis in non-HPV-expressing cell lines and primary human keratinocytes. The anti-apoptotic effect of HPV E6 expression was found to be mediated in part by upregulation of osteoprotegerin (OPG) and interleukin 6 (IL6). Purified OPG and IL6, when added to cells together, but not individually, reduced apoptosis following UV irradiation. We provide evidence that OPG and IL6 inhibit the extrinsic and intrinsic apoptotic pathways, respectively. Furthermore, we show by immunohistochemistry of HPV-typed SCC sections that IL6 protein is upregulated in HPV-positive tumors compared with HPV-negative cancers. These findings support the hypothesis that a small number of HPV-infected cells influence UV-induced apoptosis in the skin and contribute to tumorigenesis.
Non-melanoma skin cancer (NMSC) is the most frequent human cancer of Caucasian populations. Altho... more Non-melanoma skin cancer (NMSC) is the most frequent human cancer of Caucasian populations. Although the ultraviolet irradiation is a key contributor to the establishment of this keratinocyte malignancy, the infection by some types of human papillomavirus (HPV) has also been implicated in NMSC development. Cancers occur as a result of a complex series of interactions between the cancer cell and its surrounding matrix. The matrix metalloproteinases (MMPs) play a role in degrading the extracellular matrix. MMP9 is an important gelatinase involved in processes such as cell migration, invasion and metastasis. In this report, we demonstrated by EMSA experiments that the MMP9 promoter contains a binding site for the transcriptional regulator E2 of HPV8. Transient reporter gene assays showed that HPV8-E2 activated the MMP9 promoter in a dose-dependent manner in human epidermal keratinocytes. An E2 transactivation-defective mutant (I73L) as well as a DNA-binding deficient mutant (R433K) demonstrated no activation of the MMP9 promoter, suggesting that both an intact transactivation and DNA-binding domain are required for E2 activation of the MMP9-promoter. The functional role of the E2 binding site within the MMP9 promoter was also confirmed by mutating the E2 binding site. In organotypic cultures of human skin, an overexpression of MMP9 was observed in suprabasal layers of the HPV8 E2-expressing epidermis thus confirming the results of the monolayer cultures. These results demonstrate that the early gene E2 of HPV8 is able to increase the expression of MMP9 by direct activation of the MMP9-promoter.
Activation of the cell-death mediator Bak commits a cell to mitochondrial apoptosis. The initial ... more Activation of the cell-death mediator Bak commits a cell to mitochondrial apoptosis. The initial steps that govern Bak activation are poorly understood. To further clarify these pivotal events, we have investigated whether post-translational modifications of Bak impinge on its activation potential. In this study, we report that on apoptotic stimulation Bak undergoes dephosphorylation at tyrosine residue 108 (Y108), a critical event that is necessary but not sufficient for Bak activation, but is required both for early exposure of the occluded N-terminal domain and multimerisation. RNA interference (RNAi) screening identified non-receptor tyrosine phosphatases (PTPNs) required for Bak dephosphorylation and apoptotic induction through chemotherapeutic agents. Specifically, modulation of PTPN5 protein expression by siRNA and overexpression directly affected both Bak-Y108 phosphorylation and the initiation of Bak activation. We further show that MEK/ ERK signalling directly affects Bak phosphorylation through inhibition of PTPN5 to promote cell survival. We propose a model of Bak activation in which the regulation of Bak dephosphorylation constitutes the initial step in the activation process, which reveals a previously unsuspected mechanism controlling the initiation of mitochondrial apoptosis.
Expression of Axl, a receptor tyrosine kinase, is increased in cutaneous squamous cell carcinoma ... more Expression of Axl, a receptor tyrosine kinase, is increased in cutaneous squamous cell carcinoma (SCC). Examination of a series of cutaneous SCC tumors revealed positive phospho-Akt (P-Akt) staining accompanied by weak TUNEL staining in Axl-positive tumors, suggesting an anti-apoptotic role for Axl in SCC survival. The role of Axl in UV-induced apoptosis was investigated in a cutaneous SCC cell line using retroviral short hairpin RNA sequences enabling stable Axl knock-down. We show that, although Axl knock-down has no effect on cell proliferation, it sensitizes cells to UV-induced apoptosis through increased activation of the pro-apoptotic protein Bad, a change in the conformation of Bax and Bak, release of cytochrome c into the cytosol, and activation of caspases. These events are accompanied by faster Akt dephosphorylation in UV-treated Axl knockdown cells and correlate with the degree of Axl knock-down. Treatment with the pan-caspase inhibitor zVADfmk partially rescued cells from UV-induced apoptosis but did not affect Bid cleavage or cytochrome c release, suggesting that cells die via the mitochondrial-mediated pathway. Thus, Axl confers resistance of SCC cells to apoptosis and displays potential as a target for therapeutic intervention in cutaneous SCC.
It has been demonstrated previously that E7 oncogene expression of human papillomavirus (HPV) typ... more It has been demonstrated previously that E7 oncogene expression of human papillomavirus (HPV) type 8 in keratinocytes induces cell invasion and accelerated differentiation. Looking for cellular genes deregulated by HPV-8 E7, lipocalin-2 was identified as being upregulated in these cells by cDNA microarray analysis. Lipocalin-2 is known to be overexpressed in many human cancers and is implicated in the regulation of cell proliferation, differentiation and apoptosis. In this study, increased levels of lipocalin-2 were observed in extracts from HPV-8 E7-positive keratinocytes and from keratinocytes expressing E7 of HPV-1, -4, -5, -15, -20 and -38, but not of HPV-16. Similar results were obtained when measuring secreted lipocalin-2 in the supernatants of the cell cultures. Lipocalin-2 expression was associated with cell differentiation in keratinocyte monolayers and in organotypic skin cultures. It was found in the uppermost layers of HPV-5, -8, -15, -16, -20 and 38 E7-expressing but not low-risk HPV-1 and -4 E7-expressing keratinocytes. Immunohistochemical staining of HPV-positive and -negative human skin squamous cell carcinomas (SCCs) revealed lipocalin-2 expression mostly in differentiated, filaggrin-positive areas of 15 out of 17 HPV-positive and three out of nine HPV-negative SCCs. These data indicate that lipocalin-2 expression correlates with HPV positivity of cutaneous SCCs.
Human papillomaviruses (HPVs) of the genus Betapapillomavirus appear to be involved in the early ... more Human papillomaviruses (HPVs) of the genus Betapapillomavirus appear to be involved in the early stages of skin cancer development, since both the prevalence and viral load are higher in precancerous actinic keratoses than in skin cancers. Interleukin-8 (IL-8) is an inflammatory cytokine that serves to alert the surrounding tissue after UV-induced damage. We examined the effects of the E2, E6 and E7 proteins of HPV8 and the E6 proteins of various HPV genotypes on IL-8 secretion from primary keratinocytes. HPV5 and HPV8 E6 showed the highest downregulation of basal IL-8 secretion. HPV8 E6 also negatively modulated IL-8 mRNA expression and protein secretion upon UVB irradiation. The downregulation of IL-8 in actinic keratoses may weaken the response to UV-induced damage and thus favour the accumulation of UVB-induced mutations.
Recent studies strongly support a role of human papillomavirus type 8 (HPV8) in non-melanoma skin... more Recent studies strongly support a role of human papillomavirus type 8 (HPV8) in non-melanoma skin cancer development. In this study, a quantitative two-dimensional (2D) differential gene expression (DiGE) gel approach combined with mass spectrometry has been used to identify proteins that are abundantly deregulated in primary human epidermal keratinocytes expressing HPV8 sequences. Twenty six protein spots showed significant changes in the level of expression between keratinocytes expressing E7 or the complete early region (CER) of HPV8 compared to extracts from cells lacking HPV8 gene expression. No differences between HPV8 E7 alone and HPV8 CER expressing cells were observed. The 26 protein spots that were differentially expressed corresponded to 20 different proteins, of which 14 actin-associated proteins were downregulated except for calponin-2, which was the only actin-binding protein that was overexpressed. Besides changes in actin modulating proteins, an upregulation of cytokeratins (CK) 5, 6 and 14 was also noted. This study suggests that the actin and keratin cytoskeleton modulating proteins are targets for HPV8.
UVB induced DNA damage is the major aetiological agent in NMSC development, but mounting evidence... more UVB induced DNA damage is the major aetiological agent in NMSC development, but mounting evidence suggests a role for human papillomaviruses (HPV) from genus beta, including HPV 5 and HPV 8, in the development of NMSC on sun exposed body sites. We have previously shown that UVB activates Bak, an apoptogenic mitochondrial factor that, following an apoptotic stimulus, undergoes a conformational change that leads to pore formation in the mitochondrial membrane that releases apoptotic factors. The HPV E6 protein effectively inhibits UVB-induced apoptosis and targets Bak for proteolytic degradation. We have now identified the regions of the HPV5 E6 that are required to mediate Bak proteolysis and contribute toward the antiapoptotic activity of the protein. Interestingly, while wild-type HPV5 E6 does not bind or target p53 for proteolysis, we have isolated specific HPV5 E6 mutants that switch target specificity from Bak to p53 in a p53 codon 72 isoform-dependent manner. Furthermore, we demonstrate that the ability of wild-type HPV5 E6 to target Bak or specific E6 mutants to target p53 for proteolysis is not dependent on the E6-AP ubiquitin ligase.
The thymidine analogue 4-thiothymidine (S 4 TdR) is a photosensitizer for UVA radiation. The UV a... more The thymidine analogue 4-thiothymidine (S 4 TdR) is a photosensitizer for UVA radiation. The UV absorbance spectrum of S 4 TdR and its incorporation into DNA suggests that it might act synergistically with nonlethal doses of UVA to selectively kill hyperproliferative or cancerous skin cells. We show here that nontoxic concentrations of S 4 TdR combine with nonlethal doses of UVA to kill proliferating cultured skin cells. Established cell lines with a high fraction of proliferating cells were more sensitive than primary keratinocytes or fibroblasts to apoptosis induction by S 4 TdR/UVA. Although S 4 TdR plus UVA treatment induces stabilization of p53, cell death, as measured by apoptosis or clonal survival, occurs to a similar extent in both p53 wild-type and p53-null backgrounds. Furthermore, different types of human papilloma virus E6 proteins, which protect against UVBinduced apoptosis, have little effect on killing by S 4 TdR/ UVA. S 4 TdR/UVA offers a possible therapeutic intervention strategy that seems to be applicable to human papilloma virus -associated skin lesions. [Mol Cancer Ther 2007;6(9):2487 -95]
Formulations of ultra-deformable liposomes containing bleomycin (Bleosome TM ) have previously be... more Formulations of ultra-deformable liposomes containing bleomycin (Bleosome TM ) have previously been described and proposed for topical treatment of skin cancer . Entrapment of bleomycin in ultra-deformable liposomes. S. T. P. Pharm. Sci. 13,[237][238][239]. In this study, the stability of various Bleosome TM formulations was characterised and a purification process was established to isolate Bleosome TM for testing on cultures of either human cutaneous keratinocytes (NEB-1) immortalised by human papilloma virus (HPV)-type 16, or a spontaneously immortalised human squamous cell carcinoma (SCC) from a primary tumour. Bleosome TM facilitated entrapment of high concentrations of active bleomycin and samples purified by gel-filtration chromatography remained stable during 7 days of storage at 4 • C or at room temperature. Serially-diluted samples of this purified, high-strength product, 'high dose' were applied onto keratinocyte cell cultures to elucidate Bleosome TM LD 50 profiles.
Epithelial tumorigenesis has been linked to AKT up-regulation. Human papillomaviruses (HPV) cause... more Epithelial tumorigenesis has been linked to AKT up-regulation. Human papillomaviruses (HPV) cause anogenital cancers and anogenital HPV infection up-regulates AKT activity. Mounting evidence points to a role for cutaneous HPVs as etiologic factors in skin tumorigenesis. High-risk cutaneous β HPVs have been linked to carcinogenesis in immunosuppressed patients, and high-risk cutaneous HPV8 genes enhance tumorigenesis in transgenic mice. We find that, in contrast to anogenital HPVs, cutaneous HPV8 early genes down-regulate epidermal AKT activity by down-regulating AKT1 isoform levels. This down-regulation occurs before papilloma formation or tumorigenesis and leads to cutaneous differentiation changes that may weaken the epidermal squame for viral release. We find that, in viral warts (papillomas) and HPV gene-induced epidermal tumors, AKT activitycan be activated focally by up-regulation and phosphorylation of the AKT2 isoform. In squamous cell carcinomas (SCC), AKT1 down-regulation is also common, consistent with a viral influence, whereas AKT2 up-regulation is widespread. Activation of up-regulated AKT2 by serine phosphorylation associates with high-grade tumors. Our data suggest that AKT2 up-regulation is characteristic of SCC and that coincident AKT2 activation through serine phosphorylation correlates with malignancy. These findings highlight differences between the effects of anogenital and cutaneous HPV on epithelial AKT activity and furthermore show that AKT isoforms can behave differently during epidermal tumorigenesis. These findings also suggest AKT2 as a possible therapeutic tumor target in SCC.
The molecular mechanisms that underlie the development of squamous cell skin cancers (SSC) are po... more The molecular mechanisms that underlie the development of squamous cell skin cancers (SSC) are poorly understood. We have used oligonucleotide microarrays to compare the differences in cellular gene expression between a series of keratinocyte cell that mimic disease progression with the aim of identifying genes that may potentially contribute towards squamous cell carcinoma (SCC) progression in vivo, and in particular to identify markers that may serve as potential therapeutic targets for SCC treatment. Gene expression differences were corroborated by polymerase chain reaction and Western blotting. We identified Axl, a receptor tyrosine kinase with transforming potential that has also been shown to have a role in cell survival, adhesion and chemotaxis, was upregulated in vitro in SCC-derived cells compared to premalignant cells. Extending the investigation to tumour biopsies showed that the Axl protein was overexpressed in vivo in a series of SCCs.
A distinct variant of Epidermodysplasia verruciformis in a Turkish family lacking EVER1 and EVER2... more A distinct variant of Epidermodysplasia verruciformis in a Turkish family lacking EVER1 and EVER2 mutations Epidermodysplasia verruciformis (EV) is a rare genodermatosis associated with a high risk of skin cancer. This condition is characterized by an abnormal susceptibility to human papillomavirus genotypes from the genus beta. The cutaneous lesions of EV first appear in childhood and are highly polymorphic. Multiple squamous cell carcinomas (SCC) of the skin develop subsequently on sun-exposed
The human papillomavirus (HPV) type 18 E6 gene cooperates with activated Ha-ras to immortalize pr... more The human papillomavirus (HPV) type 18 E6 gene cooperates with activated Ha-ras to immortalize primary mouse cells in culture. Using a plasmid where HPV18 E6 expression is regulated by the glucocorticoid inducible MMTV LTR, we have generated immortalized cell lines in which the continued expression of E6 was necessary for maintenance of the transformed phenotype. In the absence of exogenously added hormone these cells were found to arrest in G0/G1. Furthermore, we demonstrate that the effects of E6 were essentially p53 independent and therefore define a novel function by which E6 is able to modulate cell proliferation. In addition, when the E6 dependent cells were maintained under conditions of prolonged growth arrest by the removal of E6, revertant cells were isolated which were no longer dependent on E6 expression for continued proliferation. These revertant cells were found to have acquired a mutation in the cellular gene p53, suggesting that certain p53 mutations are dominant over an E6 requirement in this assay.
Recent evidence suggests that an initial barrier to the emergence of tumours is a DNA damage resp... more Recent evidence suggests that an initial barrier to the emergence of tumours is a DNA damage response that evokes a counter-response which arrests the growth of, or eliminates, damaged cells. Early precursor lesions express markers of an activated DNA damage response in several types of tumour, with a diminishing response in more advanced cancers. An important marker of DNA damage is ATM which becomes phosphorylated (pATM) upon activation. We have investigated pATM expression patterns in cultured keratinocytes, skin explants and a spectrum of pre-malignant to malignant keratinocyte skin lesions by immunohistochemistry. We found that pATM was mainly localised to the Golgi apparatus, which contrasts with its nuclear localisation in other tissues. Upon UV irradiation there is transient formation of pATM in nuclear foci, consistent with recruitment to the sites of DNA damage. By immunohistochemistry we show pATM expression in precancerous keratinocyte lesions is greater and predominantly nuclear when compared to the invasive lesions where pATM is weaker and predominantly cytoplasmic. Our results are consistent with the hypothesis that the DNA damage response acts as a barrier to cutaneous tumour formation, but also suggests that ATM expression in skin is different compared to other tissues. This may be a consequence of the constant exposure of skin to UVR, and has implications for skin carcinogenesis.
Skin Cancer after Organ Transplantation, Jan 1, 2009
Human papillomaviruses (HPV) are small double-stranded DNA viruses that predominantly form hyperp... more Human papillomaviruses (HPV) are small double-stranded DNA viruses that predominantly form hyperproliferative lesions in mucosal and cutaneous epithelial tissues and are strictly epitheliotropic. More than 120 different types have been characterised based on DNA ...
Recent studies strongly support a role of human papillomavirus type 8 (HPV8) in non-melanoma skin... more Recent studies strongly support a role of human papillomavirus type 8 (HPV8) in non-melanoma skin cancer development. In this study, a quantitative two-dimensional (2D) differential gene expression (DiGE) gel approach combined with mass spectrometry has been used to identify proteins that are abundantly deregulated in primary human epidermal keratinocytes expressing HPV8 sequences. Twenty six protein spots showed significant changes in the level of expression between keratinocytes expressing E7 or the complete early region (CER) of HPV8 compared to extracts from cells lacking HPV8 gene expression. No differences between HPV8 E7 alone and HPV8 CER expressing cells were observed. The 26 protein spots that were differentially expressed corresponded to 20 different proteins, of which 14 actin-associated proteins were downregulated except for calponin-2, which was the only actin-binding protein that was overexpressed. Besides changes in actin modulating proteins, an upregulation of cytokeratins (CK) 5, 6 and 14 was also noted. This study suggests that the actin and keratin cytoskeleton modulating proteins are targets for HPV8.
Human papillomaviruses (HPVs) are small DNA viruses, which specifically infect keratinocytes at d... more Human papillomaviruses (HPVs) are small DNA viruses, which specifically infect keratinocytes at different body sites. The association between cutaneous squamous cell carcinoma (SCC) formation, ultraviolet (UV) irradiation and infection with a high-risk subset of cutaneous HPVs has been postulated although the underlying molecular mechanisms by which HPV may play a role in SCC development are not yet fully elucidated. Expression of the viral E6 oncoprotein has been shown to interfere with DNA damage responses and inhibit UV-induced apoptosis, suggesting HPV can contribute to early stages in tumorigenesis. However, cutaneous SCCs, in contrast to HPV-associated anogenital cancers do not harbor HPV DNA in every tumor cell. Here, we show that expression of E6 from the prototypic skin cancer-associated HPV type 5 induced the secretion of factors that were able to inhibit UV-induced apoptosis in non-HPV-expressing cell lines and primary human keratinocytes. The anti-apoptotic effect of HPV E6 expression was found to be mediated in part by upregulation of osteoprotegerin (OPG) and interleukin 6 (IL6). Purified OPG and IL6, when added to cells together, but not individually, reduced apoptosis following UV irradiation. We provide evidence that OPG and IL6 inhibit the extrinsic and intrinsic apoptotic pathways, respectively. Furthermore, we show by immunohistochemistry of HPV-typed SCC sections that IL6 protein is upregulated in HPV-positive tumors compared with HPV-negative cancers. These findings support the hypothesis that a small number of HPV-infected cells influence UV-induced apoptosis in the skin and contribute to tumorigenesis.
Non-melanoma skin cancer (NMSC) is the most frequent human cancer of Caucasian populations. Altho... more Non-melanoma skin cancer (NMSC) is the most frequent human cancer of Caucasian populations. Although the ultraviolet irradiation is a key contributor to the establishment of this keratinocyte malignancy, the infection by some types of human papillomavirus (HPV) has also been implicated in NMSC development. Cancers occur as a result of a complex series of interactions between the cancer cell and its surrounding matrix. The matrix metalloproteinases (MMPs) play a role in degrading the extracellular matrix. MMP9 is an important gelatinase involved in processes such as cell migration, invasion and metastasis. In this report, we demonstrated by EMSA experiments that the MMP9 promoter contains a binding site for the transcriptional regulator E2 of HPV8. Transient reporter gene assays showed that HPV8-E2 activated the MMP9 promoter in a dose-dependent manner in human epidermal keratinocytes. An E2 transactivation-defective mutant (I73L) as well as a DNA-binding deficient mutant (R433K) demonstrated no activation of the MMP9 promoter, suggesting that both an intact transactivation and DNA-binding domain are required for E2 activation of the MMP9-promoter. The functional role of the E2 binding site within the MMP9 promoter was also confirmed by mutating the E2 binding site. In organotypic cultures of human skin, an overexpression of MMP9 was observed in suprabasal layers of the HPV8 E2-expressing epidermis thus confirming the results of the monolayer cultures. These results demonstrate that the early gene E2 of HPV8 is able to increase the expression of MMP9 by direct activation of the MMP9-promoter.
Activation of the cell-death mediator Bak commits a cell to mitochondrial apoptosis. The initial ... more Activation of the cell-death mediator Bak commits a cell to mitochondrial apoptosis. The initial steps that govern Bak activation are poorly understood. To further clarify these pivotal events, we have investigated whether post-translational modifications of Bak impinge on its activation potential. In this study, we report that on apoptotic stimulation Bak undergoes dephosphorylation at tyrosine residue 108 (Y108), a critical event that is necessary but not sufficient for Bak activation, but is required both for early exposure of the occluded N-terminal domain and multimerisation. RNA interference (RNAi) screening identified non-receptor tyrosine phosphatases (PTPNs) required for Bak dephosphorylation and apoptotic induction through chemotherapeutic agents. Specifically, modulation of PTPN5 protein expression by siRNA and overexpression directly affected both Bak-Y108 phosphorylation and the initiation of Bak activation. We further show that MEK/ ERK signalling directly affects Bak phosphorylation through inhibition of PTPN5 to promote cell survival. We propose a model of Bak activation in which the regulation of Bak dephosphorylation constitutes the initial step in the activation process, which reveals a previously unsuspected mechanism controlling the initiation of mitochondrial apoptosis.
Expression of Axl, a receptor tyrosine kinase, is increased in cutaneous squamous cell carcinoma ... more Expression of Axl, a receptor tyrosine kinase, is increased in cutaneous squamous cell carcinoma (SCC). Examination of a series of cutaneous SCC tumors revealed positive phospho-Akt (P-Akt) staining accompanied by weak TUNEL staining in Axl-positive tumors, suggesting an anti-apoptotic role for Axl in SCC survival. The role of Axl in UV-induced apoptosis was investigated in a cutaneous SCC cell line using retroviral short hairpin RNA sequences enabling stable Axl knock-down. We show that, although Axl knock-down has no effect on cell proliferation, it sensitizes cells to UV-induced apoptosis through increased activation of the pro-apoptotic protein Bad, a change in the conformation of Bax and Bak, release of cytochrome c into the cytosol, and activation of caspases. These events are accompanied by faster Akt dephosphorylation in UV-treated Axl knockdown cells and correlate with the degree of Axl knock-down. Treatment with the pan-caspase inhibitor zVADfmk partially rescued cells from UV-induced apoptosis but did not affect Bid cleavage or cytochrome c release, suggesting that cells die via the mitochondrial-mediated pathway. Thus, Axl confers resistance of SCC cells to apoptosis and displays potential as a target for therapeutic intervention in cutaneous SCC.
It has been demonstrated previously that E7 oncogene expression of human papillomavirus (HPV) typ... more It has been demonstrated previously that E7 oncogene expression of human papillomavirus (HPV) type 8 in keratinocytes induces cell invasion and accelerated differentiation. Looking for cellular genes deregulated by HPV-8 E7, lipocalin-2 was identified as being upregulated in these cells by cDNA microarray analysis. Lipocalin-2 is known to be overexpressed in many human cancers and is implicated in the regulation of cell proliferation, differentiation and apoptosis. In this study, increased levels of lipocalin-2 were observed in extracts from HPV-8 E7-positive keratinocytes and from keratinocytes expressing E7 of HPV-1, -4, -5, -15, -20 and -38, but not of HPV-16. Similar results were obtained when measuring secreted lipocalin-2 in the supernatants of the cell cultures. Lipocalin-2 expression was associated with cell differentiation in keratinocyte monolayers and in organotypic skin cultures. It was found in the uppermost layers of HPV-5, -8, -15, -16, -20 and 38 E7-expressing but not low-risk HPV-1 and -4 E7-expressing keratinocytes. Immunohistochemical staining of HPV-positive and -negative human skin squamous cell carcinomas (SCCs) revealed lipocalin-2 expression mostly in differentiated, filaggrin-positive areas of 15 out of 17 HPV-positive and three out of nine HPV-negative SCCs. These data indicate that lipocalin-2 expression correlates with HPV positivity of cutaneous SCCs.
Human papillomaviruses (HPVs) of the genus Betapapillomavirus appear to be involved in the early ... more Human papillomaviruses (HPVs) of the genus Betapapillomavirus appear to be involved in the early stages of skin cancer development, since both the prevalence and viral load are higher in precancerous actinic keratoses than in skin cancers. Interleukin-8 (IL-8) is an inflammatory cytokine that serves to alert the surrounding tissue after UV-induced damage. We examined the effects of the E2, E6 and E7 proteins of HPV8 and the E6 proteins of various HPV genotypes on IL-8 secretion from primary keratinocytes. HPV5 and HPV8 E6 showed the highest downregulation of basal IL-8 secretion. HPV8 E6 also negatively modulated IL-8 mRNA expression and protein secretion upon UVB irradiation. The downregulation of IL-8 in actinic keratoses may weaken the response to UV-induced damage and thus favour the accumulation of UVB-induced mutations.
Recent studies strongly support a role of human papillomavirus type 8 (HPV8) in non-melanoma skin... more Recent studies strongly support a role of human papillomavirus type 8 (HPV8) in non-melanoma skin cancer development. In this study, a quantitative two-dimensional (2D) differential gene expression (DiGE) gel approach combined with mass spectrometry has been used to identify proteins that are abundantly deregulated in primary human epidermal keratinocytes expressing HPV8 sequences. Twenty six protein spots showed significant changes in the level of expression between keratinocytes expressing E7 or the complete early region (CER) of HPV8 compared to extracts from cells lacking HPV8 gene expression. No differences between HPV8 E7 alone and HPV8 CER expressing cells were observed. The 26 protein spots that were differentially expressed corresponded to 20 different proteins, of which 14 actin-associated proteins were downregulated except for calponin-2, which was the only actin-binding protein that was overexpressed. Besides changes in actin modulating proteins, an upregulation of cytokeratins (CK) 5, 6 and 14 was also noted. This study suggests that the actin and keratin cytoskeleton modulating proteins are targets for HPV8.
UVB induced DNA damage is the major aetiological agent in NMSC development, but mounting evidence... more UVB induced DNA damage is the major aetiological agent in NMSC development, but mounting evidence suggests a role for human papillomaviruses (HPV) from genus beta, including HPV 5 and HPV 8, in the development of NMSC on sun exposed body sites. We have previously shown that UVB activates Bak, an apoptogenic mitochondrial factor that, following an apoptotic stimulus, undergoes a conformational change that leads to pore formation in the mitochondrial membrane that releases apoptotic factors. The HPV E6 protein effectively inhibits UVB-induced apoptosis and targets Bak for proteolytic degradation. We have now identified the regions of the HPV5 E6 that are required to mediate Bak proteolysis and contribute toward the antiapoptotic activity of the protein. Interestingly, while wild-type HPV5 E6 does not bind or target p53 for proteolysis, we have isolated specific HPV5 E6 mutants that switch target specificity from Bak to p53 in a p53 codon 72 isoform-dependent manner. Furthermore, we demonstrate that the ability of wild-type HPV5 E6 to target Bak or specific E6 mutants to target p53 for proteolysis is not dependent on the E6-AP ubiquitin ligase.
The thymidine analogue 4-thiothymidine (S 4 TdR) is a photosensitizer for UVA radiation. The UV a... more The thymidine analogue 4-thiothymidine (S 4 TdR) is a photosensitizer for UVA radiation. The UV absorbance spectrum of S 4 TdR and its incorporation into DNA suggests that it might act synergistically with nonlethal doses of UVA to selectively kill hyperproliferative or cancerous skin cells. We show here that nontoxic concentrations of S 4 TdR combine with nonlethal doses of UVA to kill proliferating cultured skin cells. Established cell lines with a high fraction of proliferating cells were more sensitive than primary keratinocytes or fibroblasts to apoptosis induction by S 4 TdR/UVA. Although S 4 TdR plus UVA treatment induces stabilization of p53, cell death, as measured by apoptosis or clonal survival, occurs to a similar extent in both p53 wild-type and p53-null backgrounds. Furthermore, different types of human papilloma virus E6 proteins, which protect against UVBinduced apoptosis, have little effect on killing by S 4 TdR/ UVA. S 4 TdR/UVA offers a possible therapeutic intervention strategy that seems to be applicable to human papilloma virus -associated skin lesions. [Mol Cancer Ther 2007;6(9):2487 -95]
Formulations of ultra-deformable liposomes containing bleomycin (Bleosome TM ) have previously be... more Formulations of ultra-deformable liposomes containing bleomycin (Bleosome TM ) have previously been described and proposed for topical treatment of skin cancer . Entrapment of bleomycin in ultra-deformable liposomes. S. T. P. Pharm. Sci. 13,[237][238][239]. In this study, the stability of various Bleosome TM formulations was characterised and a purification process was established to isolate Bleosome TM for testing on cultures of either human cutaneous keratinocytes (NEB-1) immortalised by human papilloma virus (HPV)-type 16, or a spontaneously immortalised human squamous cell carcinoma (SCC) from a primary tumour. Bleosome TM facilitated entrapment of high concentrations of active bleomycin and samples purified by gel-filtration chromatography remained stable during 7 days of storage at 4 • C or at room temperature. Serially-diluted samples of this purified, high-strength product, 'high dose' were applied onto keratinocyte cell cultures to elucidate Bleosome TM LD 50 profiles.
Epithelial tumorigenesis has been linked to AKT up-regulation. Human papillomaviruses (HPV) cause... more Epithelial tumorigenesis has been linked to AKT up-regulation. Human papillomaviruses (HPV) cause anogenital cancers and anogenital HPV infection up-regulates AKT activity. Mounting evidence points to a role for cutaneous HPVs as etiologic factors in skin tumorigenesis. High-risk cutaneous β HPVs have been linked to carcinogenesis in immunosuppressed patients, and high-risk cutaneous HPV8 genes enhance tumorigenesis in transgenic mice. We find that, in contrast to anogenital HPVs, cutaneous HPV8 early genes down-regulate epidermal AKT activity by down-regulating AKT1 isoform levels. This down-regulation occurs before papilloma formation or tumorigenesis and leads to cutaneous differentiation changes that may weaken the epidermal squame for viral release. We find that, in viral warts (papillomas) and HPV gene-induced epidermal tumors, AKT activitycan be activated focally by up-regulation and phosphorylation of the AKT2 isoform. In squamous cell carcinomas (SCC), AKT1 down-regulation is also common, consistent with a viral influence, whereas AKT2 up-regulation is widespread. Activation of up-regulated AKT2 by serine phosphorylation associates with high-grade tumors. Our data suggest that AKT2 up-regulation is characteristic of SCC and that coincident AKT2 activation through serine phosphorylation correlates with malignancy. These findings highlight differences between the effects of anogenital and cutaneous HPV on epithelial AKT activity and furthermore show that AKT isoforms can behave differently during epidermal tumorigenesis. These findings also suggest AKT2 as a possible therapeutic tumor target in SCC.
The molecular mechanisms that underlie the development of squamous cell skin cancers (SSC) are po... more The molecular mechanisms that underlie the development of squamous cell skin cancers (SSC) are poorly understood. We have used oligonucleotide microarrays to compare the differences in cellular gene expression between a series of keratinocyte cell that mimic disease progression with the aim of identifying genes that may potentially contribute towards squamous cell carcinoma (SCC) progression in vivo, and in particular to identify markers that may serve as potential therapeutic targets for SCC treatment. Gene expression differences were corroborated by polymerase chain reaction and Western blotting. We identified Axl, a receptor tyrosine kinase with transforming potential that has also been shown to have a role in cell survival, adhesion and chemotaxis, was upregulated in vitro in SCC-derived cells compared to premalignant cells. Extending the investigation to tumour biopsies showed that the Axl protein was overexpressed in vivo in a series of SCCs.
A distinct variant of Epidermodysplasia verruciformis in a Turkish family lacking EVER1 and EVER2... more A distinct variant of Epidermodysplasia verruciformis in a Turkish family lacking EVER1 and EVER2 mutations Epidermodysplasia verruciformis (EV) is a rare genodermatosis associated with a high risk of skin cancer. This condition is characterized by an abnormal susceptibility to human papillomavirus genotypes from the genus beta. The cutaneous lesions of EV first appear in childhood and are highly polymorphic. Multiple squamous cell carcinomas (SCC) of the skin develop subsequently on sun-exposed
The human papillomavirus (HPV) type 18 E6 gene cooperates with activated Ha-ras to immortalize pr... more The human papillomavirus (HPV) type 18 E6 gene cooperates with activated Ha-ras to immortalize primary mouse cells in culture. Using a plasmid where HPV18 E6 expression is regulated by the glucocorticoid inducible MMTV LTR, we have generated immortalized cell lines in which the continued expression of E6 was necessary for maintenance of the transformed phenotype. In the absence of exogenously added hormone these cells were found to arrest in G0/G1. Furthermore, we demonstrate that the effects of E6 were essentially p53 independent and therefore define a novel function by which E6 is able to modulate cell proliferation. In addition, when the E6 dependent cells were maintained under conditions of prolonged growth arrest by the removal of E6, revertant cells were isolated which were no longer dependent on E6 expression for continued proliferation. These revertant cells were found to have acquired a mutation in the cellular gene p53, suggesting that certain p53 mutations are dominant over an E6 requirement in this assay.
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Papers by Alan Storey