Ras-transfection up-regulated HaCaT cell migration: inhibition by Marimastat.

Cell migration is an essential process in physiological and pathological conditions such as wound healing and tumor invasion. This phenomenon involves cell adhesion on the extracellular matrix mediated by integrins, and cell detachment promoted in part by metalloproteinases (MMPs). In the present study, the migration of two HaCaT-ras clones (metastatic or not), was compared with HaCaT cells, and normal human primary cultured keratinocytes. Using colloidal gold migration assay, the migration index on type I and type IV collagen was similar for primary cultured keratinocytes and HaCaT, whereas it was markedly higher for the HaCaT-ras clones. High motility of ras-transfected cells was confirmed from an in vitro wound healing assay. It was not correlated with changes in integrin expression or related to a different adhesion on extracellular matrix. The Marismastat (BB-2516), a MMP inhibitor, inhibited in a dose-dependent effect the migration in both assays, demonstrating the important role of MMPs in the migration process. Under our experimental conditions, MMP-1 activity was not detected in HaCaT and MMP-9 activity was secreted by these cells only after their stimulation by EGF. Here, MMP-2 was the major gelatinolytic activity secreted by all the cells and its secretion was markedly higher for HaCaT-nis clones compared with HaCaT. In addition, Western blotting results confirmed a higher expression of MMP-2 associated with a lower expression of TIMP-2 in HaCaT-ras compared with HaCaT. These results suggest that Ha-ras oncogene could be a stimulating factor of migration and might modified the balance between MMP-2 and TIMP-2 in keratinocyte cell lines.

A comparison of methods for the detection of human papillomavirus DNA by in situ hybridization with biotinylated probes on human carcinoma cell lines. Application to wart sections.

We compared nine different techniques for the detection of biotinylated DNA-DNA HPV hybrids on HeLa cells with 10-50 copies of HPV 18 DNA per cell. CaSki cells with 600 copies of HPV 16 DNA per cell and tissue sections from frozen or paraffin-embedded biopsy specimens. The cell samples were either cell deposits or cytocentrifuged or cultured slides. In most cases, the samples (cell deposits and tissue sections) were denatured with hybridization mixture prepared under stringent conditions (Tm = -17 degrees C) containing biotinylated DNA probes (cloned HPV types 1, 2, 6, 11, 16 and 18), at 90 degrees C for 10 min. In other cases (cytocentrifuged or cultured cells), the denaturation was performed by HCl hydrolysis and mild heating at 50 degrees C; the probes were denatured separately by heating. All the samples were further incubated overnight at 37 degrees C. For HPV DNA detection, three amplification levels were used on cell deposits. Only the techniques involving a three-step reaction (a rabbit anti-biotin antibody - a biotinylated goat anti-rabbit antibody - a complex of streptavidin-alkaline phosphatase or streptavidin-gold or streptavidin-fluorescein) gave satisfactory results, on both cell lines. With the one step reaction (an avidin-horseradish peroxidase, or streptavidin-alkaline phosphatase or streptavidin-fluorescein complex), no labeling of HeLa cells was observed with any of the HPV probes, including HPV 18. The techniques involving four steps (avidin or streptavidin - anti-avidin goat antibody or anti-streptavidin rabbit antibody - a biotinylated anti-goat (or anti-rabbit) antibody - a complex of avidin-biotin-peroxidase or streptavidin-biotin-alkaline phosphatase or streptavidin-biotin-horseradish peroxidase) resulted in high background on both cell lines. For the reproducible detection of low copy number of HPV DNA (less than 50 copies) such as occur in HeLa cells our data suggested that the three-step technique with the streptavidin-alkaline phosphatase complex was the method of choice. The most intense labeling was always obtained with cell deposits and the technique was successfully applied to frozen and paraffin-embedded tissue sections from typical warts.

Detection of human papillomavirus DNA in CaSki and HeLa cells by fluorescent in situ hybridization. Analysis by flow cytometry and confocal laser scanning microscopy.

CaSki and HeLa cell lines, isolated from human uterine carcinomas and containing integrated human papillomavirus (HPV) DNA type 16 and 18, respectively were used to evaluate the sensitivity of HPV-DNA detection on suspended cells by fluorescent in situ hybridization using flow cytometry and on corresponding cell deposits using confocal laser scanning microscopy (CLSM). HPV DNAs were detected in cell suspensions with biotinylated DNA probes and revealed with a three-step technique: a rabbit antibiotin antibody, a biotinylated goat anti-rabbit antibody and a streptavidin-fluorescein isothiocyanate complex. By flow cytometry, HPV DNA was detectable only in CaSki cells which contained about 600 copies of HPV DNA per cell. In HeLa cells, with only 20-50 copies of HPV DNA, flow cytometry could not detect HPV DNA, whereas CLSM permitted visualization of fluorescent labelling of HPV DNA hybrids. Furthermore, CLSM showed good preservation of cellular morphology and the nucleus was clearly recognizable after fluorescent in situ hybridization and counterstaining with propidium iodide. Moreover, this examination confirmed that the fluorescent foci were specifically confined to the cell nuclei.

Quantitative studies of keratin expression with the post-embedding immunogold labeling method.

Immunoreactivity of the 56.5 KD acidic (type I) keratin was localized ultrastructurally and quantified in normal human epidermis using the specific monoclonal antibody KL1 and post-embedding immunogold labeling. The protein was detected in keratin intermediate filament bundles of all suprabasal keratinocytes. Keratohyalin granules and desmosomal plaques were labeled only on the periphery, in regions where keratin filaments penetrate these structures. The 56.5 KD keratin immunoreactivity increased from the first suprabasal layer onwards and reached its maximum in the outmost spinous layer. A subsequent abrupt decrease of the specific immunogold labeling was observed in the granular layer. This low reactivity, which persisted also in the horny layer, may be partially explained by either protein degradation or masking of the antigenic sites by a filament-aggregating material occurring at these stages of keratinocyte terminal differentiation. Statistical comparison of the quantitative results obtained in various cell and tissue compartments revealed no significant differences between the background labeling levels observed in the basal layer of epidermis with KL1, a control monoclonal antibody, or the immunogold conjugate alone. Our results confirm the specificity of 56.5 KD keratin for terminally differentiating suprabasal keratinocytes and demonstrate the importance of appropriate control studies when a post-embedding immunogold labeling method is employed.

Incidence of human papillomavirus type 33 in premalignant and malignant skin lesions from organ transplant recipients.

Human papillomavirus (HPV) type 33 belongs to potentially oncogenic types in genital cancers, but its infection corresponds to an intermediate risk for progression towards malignancy. We studied by in situ hybridization with biotinylated probes the incidence of HPV 33 infection in a series of 106 skin lesions and 12 mucosal lesions from heart and renal transplant recipients, 34 skin lesions and 17 mucosal lesions from normal population. We have shown that skin lesions from both populations could harbor HPV 33. In transplant recipients, HPV 33 was identified in 12/77 premalignant and malignant lesions and one oral leukoplakia; in the normal population, HPV 33 was detected in 2/13 warts and 2/15 mucosal lesions. The analysis of in sial hybridization signal pattern of the 17 HPV 33 positive samples suggests that a strong viral DNA signal was uniformly distributed in the nuclei of positive cell foci in 11 cases and punctate signals were seen in the nuclei of dispersed cells of 6 skin biopsies. The significance of the presence of HPV 33 DNA in skin lesions is not clear; the hybridization signal pattern may be important, mainly in premalignant actinic keratodses of organ transplant recipients although other factors are most likely involved to change the epithelial environment.

Detection of human papillomavirus DNA in squamous bronchial metaplasia and squamous cell carcinomas of the lung by in situ hybridization using biotinylated probes in paraffin-embedded specimens.

We examined a series of paraffin-embedded tissue specimens from 10 cases of squamous bronchial metaplasia and 33 cases of squamous cell carcinoma of the lung for histologic characteristics and for the presence and typing of human papillomavirus (HPV) by molecular in situ hybridization with biotinylated probes types 6, 11, 16 and 18 under stringent conditions (temperature, 19 degrees C). Fourteen of these lesions (32.5%) showed typical condylomatous histologic changes. Human papillomavirus DNA was present in seven (16%) specimens. Type 6 HPV DNA was detected in one of the squamous bronchial metaplasia cases. In six of the squamous cell carcinomas cases (18%), HPV DNA was identified (type 18, three cases; type 16, one case; type 11, one case; and type 6, one case); one of the squamous cell carcinoma specimens contained both HPV types 16 and 18. Our data confirm the presence of HPV DNA in squamous metaplastic bronchial mucosa and epidermoid lung carcinoma on paraffin-embedded tissues. This suggests that an HPV infection with benign or potentially oncogenic HPV types could be associated not only with genital tumors, but also with bronchial and lung tumors. The role of HPV DNA in the process of malignancy conversion is not yet known; HPV DNA could possibly be a cocarcinogenic factor. In situ hybridization with biotinylated probes is a useful and appropriate method of retrospective analysis of HPV DNA sequences in routinely paraffin-embedded lesions. It may be used to identify patients at risk of more serious or possibly malignant progression.

c-myc and c-Ha-ras cellular oncogenes and human papillomaviruses in benign and malignant cutaneous lesions.

To analyze the role of human papillomavirus (HPV) infection and c-myc and c-Ha-ras oncogene activation in cutaneous and mucosal lesions, serial frozen sections of 47 lesions from grafted recipients and 10 biopsies from non-immunosuppressed patients were examined. HeLa, CaSki, MCF7, Colo 320 and 3T3 cells, which contain various copy numbers of HPV DNA and/or c-myc gene, were used as controls. HPV, myc and ras oncogene DNAs were not detected in normal epithelia by in situ hybridization with biotinylated DNA probes. The amplification of ras oncogene was detected in 20/57 lesions. The amplification of myc oncogene was found in 14/57 lesions, 13 of which showed both myc and ras gene amplification. c-myc and/or c-Ha-ras DNA was more frequently amplified in cutaneous squamous cell carcinomas (8/14 cases) and anogenital papillomas (4/6 cases), than in common and plantar warts (3/14 cases) or actinic keratoses (2/10 cases). HPV DNA was detected in 26/57 biopsies. Oncogene amplification was codetected with HPV DNA in 10/26 lesions, each of them containing at least one potentially oncogenic HPV type (5,16 and/or 18). The amplification was also found in 11/31 cases in the absence of HPV DNA. No significant difference was observed in the detection of HPV or oncogene DNA between the lesions of transplant recipients and those of the non-immunosuppressed population. Viral antigen was detected in 17/55 lesions by indirect immunofluorescence; 5 of the positive biopsies showed ras oncogene amplification. Myc and ras p21 oncoproteins were respectively localized in the nuclei and on the membrane of epithelial cells by indirect immunofluorescence. A good correlation was observed between the amplification of oncogenes and the expression of oncoproteins. Our results favor the hypothesis of a cooperation between HPV infection and myc and ras oncogene activation in skin carcinogenesis.

Differences of reactivity to interferon gamma in HeLa and CaSki cells: a combined immunocytochemical and flow-cytometric study.

We characterized the changes induced by treatment for 48 h with 100 U/ ml interferon gamma (IFN-gamma) on HeLa and CaSki cells, derived from human uterine carcinomas and containing human papillomavirus (HPV) type 16 and HPV type 18 respectively, by studying cell growth, cell morphology, the cell cycle and expression of epidermal growth factor (EGF) receptor, filaggrin-profilaggrin and MHC class II antigen, HLA-DR. The response of the two cell lines to IFN gamma differed in some cases. In both cell lines, the cells remained viable; cell growth was similarly inhibited as shown by cell counts. Signs of morphological changes were essentially observed in HeLa cells. The cell cycle phases, analyzed by flow cytometry were more disturbed in CaSki than in HeLa cells; the proportion of CaSki cells in S phase increased and those in G2 + M decreased. Expression of EGF receptors related to proliferation increased only in CaSki cells while expression of filaggrin-profilaggrin, a marker of differentiation, and HLA-DR, a marker of epithelial cell immune response, was enhanced in both cell lines. The presence of filaggrin-profilaggrin being unexpected in these cells, the specificity of the reaction with the monoclonal antibody AKH1 was confirmed by immunoblotting. In conclusion, our results show that the two cell lines reacted differently to IFN gamma although they are of similar origin and the different antigens studied may be useful to predict the progression of lesions infected with HPV towards malignancy or the reactivity to IFN gamma of such lesions. However, enhanced synthesis of EGF receptors is probably independent of the antiproliferative effect of IFN gamma but an increase in HLA-DR antigen expression by epithelial cells, which corresponds to an immune response favored by IFN gamma, could act synergistically with cell growth inhibition and differentiation to exclude tumoral and/or HPV-infected cells.

Human papillomavirus infection and filaggrin expression in paraffin-embedded biopsy specimens of extragenital Bowen's disease and genital bowenoid papulosis.

Cutaneous Bowen's disease (BD) and genital bowenoid papulosis (BP) are considered as precancerous or cancerous lesions that are sometimes infected with human papillomavirus (HPV). We studied retrospectively paraffin-embedded sections of 11 samples of cutaneous BD and 6 samples of genital BP from the general population for HPV infection and filaggrin expression. Using in situ hybridization with biotinylated probes of HPV types 1, 2, 5, 6, 11, 16, and 18, under stringent and/or non-stringent conditions and a streptavidin-alkaline-phosphatase complex for hybrid detection, HPV DNA was detected in 6/17 cases (5 BD and 1 BP). Positive nuclei were located in intermediate or upper epithelial cell layers. HPV 16 was found in 2 cases of BD but associated either with HPV 2 or 18. Three additional lesions reacted only under non-stringent conditions; HPV could not be typed with the probes used. The positive case of BP reacted with the four probe types 1, 2, 16, 18 and was negative with HPV 6 or 11. Viral antigen was not detected by indirect immunofluorescence with a rabbit antiserum directed to group-specific viral capsid antigen. Differentiation disorders were observed in the intermediate and upper cell layers of these specimens, as shown by a reduced expression of filaggrin/profilaggrin, a marker of terminal differentiation, in extragenital BD (7/11 cases), and an increased expression in genital BP (4/5 cases) although viral DNA was not always detectable. This study shows that in situ hybridization is a valuable technique for HPV DNA detection and its typing in BD and BP lesions on deparaffinized sections. The positive nuclei were located in the cell layers that exhibited abnormal expression of differentiation. There is no relation between the HPV infecting type and the filaggrin expression.

Heterogeneous immunoreactivity of frozen human benign and malignant breast lesions to C-MYC and C-Ha-ras cellular oncogenes.

C-myc and c-Ha-ras oncoprotein expression was studied by immunohistochemistry and gene detection by in situ hybridization on serial frozen sections of 32 breast lesions (19 benign biopsies and 13 infiltrating carcinomas). C-myc protein was expressed in 15/19 benign and 12/13 malignant lesions; c-myc gene was detected in 17/19 benign and 13/13 malignant lesions. Although a higher proportion of benign biopsies (8/9) showed more than 50% of protein-positive cells than malignant specimens, this cannot predict the outcome of a lesion. Conversely, p21 ras protein was expressed only in 2/19 benign lesions and in most cases of grade I to III carcinomas. The c-Ha-ras gene was always detected in a small percentage of cells, in both benign and malignant lesions. The results obtained with atypical hyperplasia, a doubtful proliferating lesion, suggests that p21 c-Ha-ras protein expression is not restricted to breast carcinomas. Although Southern blot is commonly considered as a very sensitive technique for oncogene analysis, no amplification of c-myc and c-Ha-ras gene has been demonstrated either in benign or malignant lesions. The detection, on serial frozen sections, of proteins and DNA of c-myc and c-Ha-ras, showed a possible amplification of the c-myc and c-Ha-ras genes in various benign and malignant lesions.

Detection of low copy numbers of HPV DNA by fluorescent in situ hybridization combined with confocal microscopy as an alternative to in situ polymerase chain reaction.

In genital lesions infected by human papillomavirus (HPV), histological criteria and HPV DNA typing are of prognostic value. Therefore, non-radioactive methods such as in situ hybridization are used extensively since they preserve the histological organization of the tissue, and allow the detection and characterization of HPV DNA. However, the sensitivity of these methods is often limited to detection of low copy numbers of HPV DNA in isolated cells or in tissue sections, and therefore alternative techniques have been explored. In the present study, 1-2 copies of HPV DNA were visualized in SiHa cells either by in situ amplification of nucleic acid sequences with the polymerase chain reaction (PCR) or by fluorescent in situ hybridization (FISH) associated with observation by laser scanning confocal microscopy (LSCM). The latter procedure was evaluated for use on histological tissue sections to identify low copy numbers of HPV DNA. Genital lesions which were negative by enzymatic in situ hybridization and FISH but histologically suspected of HPV infection were investigated, and intense signals were obtained both with in situ PCR and with the combined use of FISH and LSCM. Therefore, the combination of FISH with LSCM examination may be as valuable as in situ PCR to detect viral genes present in small amounts in isolated cells and in tissue sections.

Differences in reactivity to cyclosporin A and interferon-gamma of normal and HPV-transformed keratinocytes.

In humans, cyclosporin A (CsA) avoids organ allograft rejection but induces skin carcinomas after long term immunosuppressive treatment; some of these lesions contain human papillomavirus (HPV) DNA. Interferon-gamma (IFN-gamma) is sometimes used in local treatment of persistent or recurrent lesions in normal population. In vivo, both drugs have an effect on keratinocytes which remains unclear. Therefore, their effect was studied on in vitro models of normal or HPV-transformed epithelial cell cultures. After exposure of proliferating cells for 1-3 days to 0.5-16 micrograms/ml CsA and 5-160 U/ml IFN-gamma, no cytotoxicity was observed; cell growth was inhibited; cell morphology was altered with CsA and cytoplasmic vacuoles were seen in some cells. Changes in the cell cycle were mainly obtained after treatment with 8 micrograms/ml CsA or 160 U/ml IFN-gamma, with an accumulation in S-phase especially in HPV-transformed cells. Thus, CsA and IFN-gamma affected, normal and HPV-transformed epithelial cells, differently.

The up-regulation of vascular endothelial growth factor in mutated Ha-ras HaCaT cell lines is reduced by a farnesyl transferase inhibitor.

The induction of tumor angiogenesis is mediated in particular by an increased production of VEGF. As ras oncogene is implicated in tumorigenesis, the inhibition of farnesyl transferase activity has recently been developed. The purpose of this study was to evaluate whether expression of mutated Ha-ras oncogene is associated with an altered expression of VEGF in an in vitro model of human skin carcinogenesis and to appreciate the effect of a new farnesyl transferase inhibitor on this VEGF expression. The amounts of VEGF secreted by an HaCaT cell line and two cell clones (metastatic or not) obtained after mutated c-Ha-ras transfection were compared. Our findings showed that the release of VEGF is greater for HaCaT-ras than for HaCaT cells and could be down-regulated using a protein farnesyl transferase inhibitor, in a reversible and dose-dependent manner. These results confirm that the Ha-ras oncogene can contribute to tumor development and progression of epidermal tumors through neoangiogenesis and that farnesyl transferase inhibitors as anticancer drugs may be efficient for the reduction of skin tumor growth.

Detection of herpes simplex virus DNA in a cutaneous squamous cell carcinoma by in situ hybridization.

A case of a cutaneous squamous cell carcinoma (SCC) occurring at the site of a long-standing recurrent HSV infection is described. No deficit of the cell-mediated immunity was recorded. HSV2 was isolated in several viral cultures. HSV DNA was visualized in the SCC by in situ hybridization with biotinylated probes in paraffin-embedded tissue. The samples were negative for HPV2, 5, 16, and 18 probes. A causal relationship between HSV infection and cutaneous SCC is hypothesized.

Filaggrin expression in cutaneous and mucosal human papillomavirus induced lesions.

A series of 32 human papillomavirus induced lesions derived from epidermis and mucosa was studied for the modulation of filaggrin-profilaggrin (F-PF) expression according to the degree of virus infection as compared to normal skin and mucosa biopsies. This investigation was carried out on frozen sections using indirect immunofluorescence for filaggrin detection and group specific viral antigen and by in situ hybridization with biotinylated probes for viral DNA detection and typing. The 9 cutaneous warts showed an increase of F-PF expression in upper layer cells as compared to normal epidermis, which could be related to the high production of virus (viral antigen and HPV types 1 or 2). The 5 condyloma acuminata displayed also an enhanced expression of these components which was located in several upper layers but virus infection was confirmed in 2 of them with HPV types 6, 11 or 16. The 6 laryngeal papillomas exhibited a granular reactivity pattern for F-PF in suprabasal cell layers with an increase in the upper layers; viral antigen was found in 4 cases and HPV DNA types 6, 11 or 16 were detected in 4 specimens. Conversely among 12 cervical intraepithelial neoplasia, F-PF was expressed only in very superficial layers in few cases, without any correlation with the DNA detection (6, 11 or 16, 18). Taken together these data are suggestive of an intense expression of F-PF in benign lesions which can replicate the virus and a discrete or an absent expression of these components in premalignant or malignant lesions.

Influence of fixation on human papillomavirus DNA detection in frozen and embedded paraffin lesions by in situ hybridization with biotinylated probes.

Series of frozen or paraffin-embedded tissues from various body sites, taken from non-immunosuppressed or immunosuppressed patients with persistent papilloma lesions were examined for the presence of group specific antigen from human papillomavirus (HPV) by indirect immunofluorescence or HPV DNA by in situ hybridization with biotinylated probes. We have shown that it is possible to detect HPV DNA after fixation of tissues in neutral formalin, Bouin's or Baker's solution. However, the sensitivity was reduced as compared to frozen tissues. The HPV DNA was detected in nuclei of heavily infected epithelial cells such as plantar or hand warts or in dispersed cells containing high copy numbers of HPV DNA from lesions such as squamous cell carcinomas or keratoacanthomas. In premalignant or malignant lesions of both immunosuppressed or non-immunosuppressed patients, HPV DNA was rarely detected after fixation. HPV types commonly described for skin and genital samples were identified in non-immunosuppressed patients, whereas in transplant recipients oncogenic HPV type 16 was identified in benign warts as well as in premalignant or malignant lesions. Positive reactions with several HPV types were more frequent in lesions from grafted patients than from the normal population. Virus antigen was detectable more frequently in frozen sections than in fixed tissues. Our findings indicate that in situ hybridization is an appropriate and rapid technique to study the presence of HPV infection. However, numerous controls are needed to avoid misinterpretations.

Human papillomavirus DNA in cervix. In-situ hybridization with biotinylated probes on Bouin's fixed paraffin embedded specimens.

We examined retrospectively a series of 65 Bouin's fixed, paraffin-embedded tissue specimens from 8 condylomatous lesions, 16 condylomas associated with cervical intraepithelial neoplasia (CIN), and 12 neoplasia without condylomatous signs, for histological characteristics, the detection of viral structural antigen, the presence and typing of HPV DNA by molecular in situ hybridization with biotinylated probes types 6, 11, 16 and 18 under stringent conditions (Tm - 12 degrees C). HPV DNA was present in 34/65 (52%) specimens. Detection of viral structural antigen was positive in only 14% (3/22) specimens. HPV DNA were identified in 9/9 (100%) condylomatous lesions (with HPV type 6, 11, 18). Three condylomas were coinfected with both HPV type 6 or 11 and type 18; viral antigen was found in two specimens. HPV DNA were detected in 18/31 (58%) low grade and advanced CIN associated with condylomatous changes (type 6 = 5 specimens, type 11 = 3 specimens, type 16 = 4 specimens, type 18 = 6 specimens). Four of these cases were coinfected with both HPV type 6/11 and HPV type 16/18. Viral antigen was negative in all specimens. HPV DNA were detected in 7/25 (28%) advanced intra-cervical neoplasia (CIN III) without anatomopathological condylomatous changes (type 6 = 1 specimen, type 16 = 3 specimens, type 18 = 3 specimens). One of these specimens contained both HPV types 6 and 18. Viral antigen was found in one case. Our data confirm the association of HPV types 6 and 11 with condyloma and low grade neoplasia; HPV types 16 and 18 were associated with advanced cervical neoplasia.(ABSTRACT TRUNCATED AT 250 WORDS)

Papilloma viruses, warts, carcinoma and Langerhans cells.

In human papillomavirus (HPV) infections, Langerhans cells (LC) are essential in the control of viral infection. The evolution of HPV-derived lesions in the normal population and in graft patients is drastically different, since a high proportion of papillomas progress towards malignancy in transplant recipients. We analyzed the distribution of markers of LC and T lymphocytes, the level of keratinocyte activation and the prevalence of HPV in a series of epithelial lesions obtained from the normal population and from graft patients. The local immune response of warts, condyloma acuminata, Bowen, basal and squamous cell carcinomas (SCC) showed a moderate to intense inflammatory reaction of HLA-DR positive cells, the intensity of the immune reaction being correlated with the degree of malignancy. In the normal population, CD4-positive cells were mainly overexpressed in the dermal infiltrate of condyloma and malignant lesions, whereas in grafted patients such infiltrates were CD4- and CD8-positive without significant predominance of a single T cell subset. The epidermis of most lesions was characterized by a reduced number of CD1a-positive LC with an altered morphology. This was concomitant with the decrease or loss of beta 2-microglobulin by epithelial cells. HLA-DR antigen was sometimes expressed by keratinocytes in genital lesions and SCC from the normal population but has not been detected in immunosuppressed patients. Whereas in the normal population HPV infection was only detected in benign papillomas, both benign and oncogenic HPV DNA may be present in carcinomas from graft patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Langerhans cells, inflammation markers and human papillomavirus infections in benign and malignant epithelial tumors from transplant recipients.

Organ transplant recipients frequently develop warts which progress toward premalignant or malignant lesions after a rather long grafting period. The local immune responses of such lesions (warts, condyloma acuminata, actinic keratoses, Bowen, basal and squamous cell carcinomas) was studied in 32 frozen skin specimens taken from 15 male transplant recipients and compared to similar lesions from the normal population. We studied the expression of T cell subsets, Langerhans cell phenotype, HLA class 1 (beta 2-microglobulin), HLA class 2 (DR antigen), and intercellular adhesion molecule 1 (ICAM 1). The presence of HPV infection was also considered, using in situ hybridization with biotinylated probes in order to examine the correlation with immunological markers. In the dermis, the lesions from grafted patients showed a moderate to intense inflammatory reaction of HLA-DR-positive cells. Most of these cells were CD4+ and CD8+ without any predominance of a single T cell subset. In the epidermis, most lesions were characterized by a reduced number of CD1-positive cells; this was concomitant with a decrease or a loss of beta 2-microglobulin expression by epithelial cells. HLA-DR antigen was not expressed by keratinocytes or tumoral cells in any specimen; ICAM 1 antigen was observed in a few cases. The expression of these markers was similarly modified with or without the presence of HPV DNA. Conversely, most lesions from non-immunocompromised patients, except warts, showed intense inflammatory reactions, with a predominance of CD4-positive cells and large foci of ICAM 1-positive cells. Expression of activation markers by keratinocytes occurred mainly in condylomas and squamous cell carcinomas. In the normal population, HPV infection was only detected in papilloma lesions. These data indicate, in lesions from grafted patients, a lack of effective immune response with partial inhibition of activation markers expressed by keratinocytes. It is conceivable that immunosuppressive treatment with solar exposure may also be responsible for the local immune deficiency and thus for the conversion of benign warts toward malignant lesions in grafted patients.

[Human papillomaviruses and cellular oncogenes (c-myc, c-Ha-ras) in cutaneous and mucosal lesions in transplantation recipients]. / Papillomavirus humains et oncogènes cellulaires (c-myc, c-Ha-ras) dans les lésions cutanées et muqueuses des sujets transplantés.

Transplant recipients develop numerous benign and malignant cutaneous and mucosal lesions in which histological signs of human papillomavirus (HPV) infection are observed. To investigate the role of HPV and c-myc and c-Ha-ras cellular oncogenes' activation in transplanted patients lesions, we used in situ hybridization with biotinylated probes and Southern blot to detect HPV and oncogenes DNA. We analyzed 36 lesions from grafted patients: 11 common warts, 10 actinic keratoses, 13 squamous cell carcinomas and 2 anogenital papillomas. With in situ hybridization, HPV DNA was detected in 14/36 lesions, 10 of which contained several HPV types. Benign and potentially oncogenic HPV types were detected in warts as well as in squamous cell carcinomas. The Southern blot technique confirmed the distribution of HPV types. Group specific viral antigen was detected in 12 lesions, mainly warts. C-Ha-ras oncogene was amplified in 13 lesions and c-myc oncogene in 10 lesions, 9 of which showed both oncogene amplification. The results obtained with in situ hybridization for c-myc gene amplification were confirmed with the Southern blot technique in 11/14 cases. Ras and/or myc amplification was more frequent in squamous cell carcinomas and anogenital papillomas than in warts and actinic keratoses. The amplification was not always linked to the presence of HPV DNA; however, it was more frequent in lesions infected by potentially oncogenic HPV types than in lesions containing only benign HPV types. Myc and p21 oncoproteins were respectively localized in the nucleus and on the membrane of epithelial cells by immunofluorescence. Most lesions showed a good concordance between the detection of oncogene DNA and proteins. Our results suggest than c-Ha-ras and c-myc cellular oncogenes' activation and HPV infection could play a role in the cancerization of cutaneous lesions from transplant recipients.