transforming-growth-factor-beta and Papilloma

The multistage evolution of squamous cell cancer on mouse skin has provided a model to dissect the biological and genetic changes that contribute to specific stages of carcinogenesis. Keratinocyte cell culture models have been developed that reproduce the genetic and epigenetic events in multistage skin carcinogenesis, and these provide insights into the biochemistry of the process. When the v-rasHa oncogene is transduced into normal mouse keratinocytes, the resultant papilloma phenotype is characterized by a high rate of cell proliferation, an aberrant program of differentiation marker expression, and resistance to terminal differentiation. These changes are attributed to differential effects on isoforms of protein kinase C coupled to activation of the epidermal growth factor receptor. Premalignant progression requires additional genetic changes in the v-rasHa-transduced cells. The acquisition of these changes is suppressed by transforming growth factor beta (TGF beta), and the absence of TGF beta in premalignant tumors indicates a high risk for malignant progression. Keratinocytes that are genetically null for the TGF beta 1 gene rapidly progress to squamous cell carcinomas when transduced with the v-rasHa oncogene. In culture, TGF beta 1 null keratinocytes exhibit a high rate of gene amplification that can be suppressed by concentrations of exogenous TGF beta well below those required to inhibit proliferation. This model is well-suited to identifying critical genetic changes that contribute to premalignant progression.

Topics: Animals; Calcium; Humans; Mice; Papilloma; Precancerous Conditions; RNA, Messenger; Skin Neoplasms; Transforming Growth Factor beta

Recurrent respiratory papillomatosis (RRP) is a human papillomavirus (HPV) driven neoplastic disorder of the upper aerodigestive tract that causes significant morbidity and can lead to fatal airway obstruction. Prior clinical study demonstrated clinical benefit with the programmed death-ligand 1 (PD-L1) monoclonal antibody avelumab. Bintrafusp alpha is a bifunctional inhibitor of PD-L1 and transforming growth factor-beta (TGF-b) that has shown clinical activity in several cancer types.. We conducted a phase II clinical trial evaluating bintrafusp alpha in adults with RRP. Papilloma samples before and after treatment with bintrafusp alpha were assessed for correlates of response with multiplex immunofluorescence as well as immunological and genomic analyses. Post hoc analyses of papilloma samples before and after treatment with avelumab were assessed for comparison.. Dual PD-L1/TGF-b inhibition failed to abrogate papilloma growth in most subjects and increased the frequency of clinically indicated interventions after treatment in four of eight subjects based on each subject's own historical control. TGF-b neutralization consistently decreased pSMAD3 and p21 and increased Ki67 expression within the basal layers of papillomas, indicating that TGF-b restrained proliferation. These alterations were not observed in papillomas treated with PD-L1 blockade alone. Dual PD-L1/TGF-b inhibition did not enhance anti-HPV immunity within papillomas beyond that observed with PD-L1 blockade. Genomic alterations in TGF-b superfamily genes were infrequent in papillomas and normal mucosa but present in a significant fraction of head and neck carcinomas.. Intact TGF-b signaling restrains proliferation within papillomas, and the use of clinical agents that abrogate this pathway should be avoided in patients with RRP.. NCT03707587 and NCT02859454.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Female; Humans; Immune Checkpoint Inhibitors; Immunologic Factors; Immunotherapy; Mice; NIH 3T3 Cells; Papilloma; Papillomavirus Infections; Respiratory Tract Infections; Transforming Growth Factor beta; Tumor Microenvironment

The role of natural killer (NK) cells in juvenile-onset recurrent respiratory papillomatosis (JORRP) patients remains elusive. In this study, we find increased NK cell percentage, particularly CD11b-CD27- (DN) subsets in peripheral blood of JORRP patients and associated with disease activity. RNA sequencing shows a downregulated "natural killer cell-mediated cytotoxicity" feature in JORRP tumors. We also find impaired cytotoxic capacity and lower expression of NK cell-activating receptors including NKp30 and NKp46. Higher transforming growth factor-beta 1 (TGF-β1) is found both in plasma and tumor tissues of JORRP, and anti-TGF-β1 antibody could restore NK cell cytolytic activity and upregulate NKp30 and NKG2D expression. Also, we find a significantly higher Chemokine receptor type 6 (CXCR6) on NK cells in tumors compared with that in peripheral blood. Finally, RT-PCR analysis show that both HPV6-E6-E7 and HPV11-E6-E7 overexpression leads to higher

Topics: Humans; Killer Cells, Natural; Papilloma; Papillomavirus Infections; Receptors, Natural Killer Cell; Respiratory Tract Infections; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Disease Progression; Human papillomavirus 11; Humans; Intercellular Adhesion Molecule-1; Interleukin-10; Interleukin-4; Killer Cells, Natural; Natural Cytotoxicity Triggering Receptor 3; Neoplasm Recurrence, Local; Papilloma; Papillomavirus Infections; Respiratory Tract Infections; Transforming Growth Factor beta

Chloride intracellular channel (CLIC) 4 is a member of a redox-regulated, metamorphic multifunctional protein family, first characterized as intracellular chloride channels. Current knowledge indicates that CLICs participate in signaling, cytoskeleton integrity and differentiation functions of multiple tissues. In metabolically stressed skin keratinocytes, cytoplasmic CLIC4 is S-nitrosylated and translocates to the nucleus where it enhances transforming growth factor-β (TGF-β) signaling by protecting phospho-Smad 2 and 3 from dephosphorylation. CLIC4 expression is diminished in multiple human epithelial cancers, and the protein is excluded from the nucleus. We now show that CLIC4 expression is reduced in chemically induced mouse skin papillomas, mouse and human squamous carcinomas and squamous cancer cell lines, and the protein is excluded from the nucleus. The extent of reduction in CLIC4 coincides with progression of squamous tumors from benign to malignant. Inhibiting antioxidant defense in tumor cells increases S-nitrosylation and nuclear translocation of CLIC4. Adenoviral-mediated reconstitution of nuclear CLIC4 in squamous cancer cells enhances TGF-β-dependent transcriptional activity and inhibits growth. Adenoviral targeting of CLIC4 to the nucleus of tumor cells in orthografts inhibits tumor growth, whereas elevation of CLIC4 in transgenic epidermis reduces de novo chemically induced skin tumor formation. In parallel, overexpression of exogenous CLIC4 in squamous tumor orthografts suppresses tumor growth and enhances TGF-β signaling. These results indicate that CLIC4 suppresses the growth of squamous cancers, that reduced CLIC4 expression and nuclear residence detected in cancer cells is associated with the altered redox state of tumor cells and the absence of detectable nuclear CLIC4 in cancers contributes to TGF-β resistance and enhances tumor development.

Topics: Animals; Cell Line, Tumor; Cell Nucleus; Cell Transformation, Neoplastic; Chloride Channels; Fibroblasts; Humans; Keratinocytes; Male; Mice; Mice, Inbred BALB C; Mice, Inbred SENCAR; Mitochondrial Proteins; Neoplasms, Squamous Cell; Oxidation-Reduction; Papilloma; Protein Transport; Signal Transduction; Skin Neoplasms; Transforming Growth Factor beta; Tumor Suppressor Proteins

Aminoacyl-transfer ribonucleic acid (tRNA) synthetases-interacting multifunctional protein (AIMP) 2 is a factor associated with the macromolecular protein synthesis machinery consisting of nine different aminoacyl-tRNA synthetases and three non-enzymatic factors. However, it was shown to work as a multifaceted regulator through the versatile interactions with diverse signal mediators. For instance, it can mediate pro-apoptotic response to DNA damage and tumor necrosis factor-alpha (TNF-alpha) stimulus and growth-arresting signal by transforming growth factor (TGF)-beta. Considering that these pathways are critically implicated in the control of tumorigenesis, AIMP2 is expected to work as a potent tumor suppressor with broad coverage against different cancer types. Here we investigated whether AIMP2 would give gene dosage effect on its pro-apoptotic and anti-proliferative activities using the wild-type, hetero- and homozygous AIMP2 cells and whether AIMP2 would be critical in preventing tumorigenesis using different in vivo tumor models. Both the apoptotic responses to DNA damage and TNF-alpha and sensitivity to growth arresting TGF-beta signal were reduced in AIMP2 hetero- and homozygous cells compared with the wild-type cells in dose-dependent manner. In all the in vivo carcinogenesis experiments, reduction of AIMP2 level in heterozygous AIMP2 mice provided higher susceptibility to tumor formation. Thus, this work proves the functional significance of AIMP2 in determination of cell proliferation and death, and as a haploinsufficient tumor suppressor.

Topics: Amino Acyl-tRNA Synthetases; Animals; Apoptosis; Cells, Cultured; Colonic Neoplasms; Disease Susceptibility; DNA Damage; Heterozygote; Lung Neoplasms; Mice; Mice, Inbred C57BL; Neoplasms; NF-kappa B; Papilloma; Skin Neoplasms; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Tumor Suppressor Proteins

TGFbetas are thought to have tumor suppressor activity in many organ systems, but receptor inactivation in mouse models has not previously resulted in increased spontaneous tumorigenesis. A study in this issue of Cancer Cell shows that mice with a targeted knockout of the type II TGFbeta receptor in stratified epithelia specifically develop spontaneous squamous cell carcinomas in the anogenital region, but not in the skin. Loss of TGFbeta signaling appears to destabilize the epithelium such that homeostasis fails in the face of persistent proliferative challenge, a normal feature of the anogenital site, and latent invasive and migratory phenotypes are unmasked.

Topics: Animals; Anus Neoplasms; Apoptosis; Carcinoma, Squamous Cell; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Epithelial Cells; Extracellular Matrix; Focal Adhesion Protein-Tyrosine Kinases; Homeostasis; Humans; Integrins; Keratin-14; Keratinocytes; Mice; Mice, Knockout; Mutation; Neoplasm Invasiveness; Papilloma; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; ras Proteins; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Skin; Skin Neoplasms; src-Family Kinases; Time Factors; Transforming Growth Factor beta; Urogenital Neoplasms; Wound Healing

Although TGFbeta is a potent inhibitor of proliferation, epithelia lacking the essential receptor (TbetaRII) for TGFbeta signaling display normal tissue homeostasis. By studying asymptomatic TbetaRII-deficient stratified epithelia, we show that tissue homeostasis is maintained by balancing hyperproliferation with elevated apoptosis. Moreover, rectal and genital epithelia, which are naturally proliferative, develop spontaneous squamous cell carcinomas with age when TbetaRII is absent. This progression is associated with a reduction in apoptosis and can be accelerated in phenotypically normal epidermis by oncogenic mutations in Ras. We show that TbetaRII deficiency leads to enhanced keratinocyte motility and integrin-FAK-Src signaling. Together, these mechanisms provide a molecular framework to account for many of the characteristics of TbetaRII-deficient invasive SQCCs.

Topics: Animals; Anus Neoplasms; Apoptosis; Carcinoma, Squamous Cell; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Epithelial Cells; Extracellular Matrix; Focal Adhesion Protein-Tyrosine Kinases; Homeostasis; Humans; Integrins; Keratin-14; Keratinocytes; Male; Mice; Mice, Knockout; Mutation; Neoplasm Invasiveness; Papilloma; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; ras Proteins; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Skin; Skin Neoplasms; src-Family Kinases; Time Factors; Transforming Growth Factor beta; Urogenital Neoplasms; Wound Healing

Transforming growth factor-beta (TGF-beta) plays a complex role in skin carcinogenesis, acting as a suppressor early in tumor development but later as a promoter. Smad proteins are important intracellular mediators of TGF-beta signaling. To determine the effect of disrupting Smad genes and TGF-beta signaling on chemically induced skin carcinogenesis in mice, transgenic mice heterozygous for Smad2 or Smad3 deletions and wild-type controls were treated with topical dimethylbenzanthracene for 7 months. Tumor multiplicity, type, and degree of differentiation were assessed by histopathology and immunohistochemistry. Smad3(+/-) mice developed significantly fewer tumors than the wild-type group (P < 0.05). This indicated a possible oncogenic function for Smad3 in skin carcinogenesis. Smad2(+/-) mice formed less-differentiated tumors than their wild-type counterparts, supporting a tumor suppressor role for Smad2. There was a significant difference (P < 0.05) in tumor type between Smad2(+/-) and Smad3(+/-) groups, suggesting that Smad2 and Smad3 may regulate different targets.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Carcinoma, Squamous Cell; Disease Models, Animal; DNA-Binding Proteins; Histological Techniques; Immunohistochemistry; Mice; Mice, Transgenic; Papilloma; Signal Transduction; Skin Neoplasms; Smad2 Protein; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta

Transforming growth factor beta (TGF-beta) is a growth-inhibitory cytokine for epithelial cells. In the mouse multistage skin carcinogenesis model, defects in TGF-beta 1 signaling reduce senescence in vitro and accelerate malignant progression in vivo. However, the precise postreceptor signaling pathways and specific roles played by Smad proteins in this process have not been defined. Here we show that senescence of v-ras(Ha)-transduced Smad3 null keratinocytes is delayed, whereas overexpression of Smad3, but not Smad2 or Smad4, induced senescence. The TGF-beta 1 target genes c-myc and p15(ink4b) were deregulated in the absence of Smad3. When transplanted to a graft site on nude mice, the v-ras(Ha)-transduced Smad3 null keratinocytes underwent rapid conversion from benign papilloma to malignant carcinoma, whereas wild-type keratinocytes predominantly formed papillomas. These results link Smad3-mediated regulation of growth control genes to senescence in vitro and tumor suppression in vivo.

Topics: Animals; Base Sequence; Carcinoma; Cell Transformation, Neoplastic; Cells, Cultured; Cellular Senescence; DNA Primers; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Keratinocytes; Mice; Mice, Nude; Papilloma; Promoter Regions, Genetic; Signal Transduction; Skin Neoplasms; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta

Endoglin is an integral membrane glycoprotein primarily expressed in the vascular endothelium, but also found on macrophages and stromal cells. It binds several members of the transforming growth factor (TGF)-beta family of growth factors and modulates TGF-beta(1)-dependent cellular responses. However, it lacks cytoplasmic signaling motifs and is considered as an auxiliary receptor for TGF-beta. We show here that endoglin is expressed in mouse and human epidermis and in skin appendages, such as hair follicles and sweat glands, as determined by immunohistochemistry. In normal interfollicular epidermis, endoglin was restricted to basal keratinocytes and absent in differentiating cells of suprabasal layers. Follicular expression of endoglin was high in hair bulb keratinocytes, but decreased in parts distal from the bulb. To address the role of endoglin in skin carcinogenesis in vivo, Endoglin heterozygous mice were subjected to long-term chemical carcinogenesis treatment. Reduction in endoglin had a dual effect during multistage carcinogenesis, by inhibiting the early appearance of benign papillomas, but increasing malignant progression to highly undifferentiated carcinomas. Our results are strikingly similar to those previously reported for transgenic mice overexpressing TGF-beta(1) in the epidermis. These data suggest that endoglin might attenuate TGF-beta(1) signaling in normal epidermis and interfere with progression of skin carcinogenesis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Antigens, CD; Carcinoma; Cells, Cultured; Endoglin; Epidermal Cells; Epidermis; Humans; Keratinocytes; Mice; Mice, Mutant Strains; Neoplasms, Experimental; Papilloma; Receptors, Cell Surface; Reference Values; Signal Transduction; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta; Vascular Cell Adhesion Molecule-1

Smad7 and Smad6 are inhibitory Smads that block transforming growth factor-beta (TGF-beta) superfamily signal transduction. Smad7 is overexpressed in chemically induced mouse epidermal tumors, where oncogenic activation of c-ras is a frequent event. To test the role of Smad7 overexpression in tumor progression, we used retroviruses to transduce Smad7 or Smad6 and v-ras(Ha) into primary mouse keratinocytes. By itself, Smad7 transiently enhanced keratinocyte proliferation, blocked normal differentiation, and induced keratin 8, a marker of malignant conversion, but did not cause tumor formation. Smad7 extended the in vitro life span, suppressed senescence, and increased transformation frequency 3-fold of primary keratinocytes coexpressing v-ras(Ha). Smad7/v-ras(Ha) coinfected keratinocytes rapidly progressed to squamous cell carcinomas in vivo, whereas pBabe/v-ras(Ha)- or Smad6/v-ras(Ha)-transduced keratinocytes formed only benign papillomas. Smad7/v-ras(Ha) tumors had elevated proliferation and defective nuclear localizaton of Smad2, Smad3, and Smad5, whereas only Smad5 was altered in Smad6/v-ras(Ha) tumors. Smad7 overexpression in vitro induced epidermal growth factor (EGF)-like growth factors TGF-alpha, heparin binding-EGF, amphiregulin, and EGF receptor tyrosine phosphorylation as well as the EGF-CFC growth factor cripto-1. TGF-alpha and cripto-1 were also overexpressed in Smad7/v-ras(Ha) tumors. These results suggest that Smad7 overexpression accelerates tumor progression through inhibition of TGF-beta superfamily signaling and up-regulation of the EGF-like superfamily of growth factors. This is the first demonstration that Smad7 overexpression can cause malignant conversion in a multistage cancer model and suggests that it may have an important role in the pathogenesis of human cancer.

Topics: Animals; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Disease Models, Animal; DNA-Binding Proteins; Genes, ras; Keratinocytes; Mice; Mice, Inbred BALB C; Mice, Nude; NIH 3T3 Cells; Papilloma; Retroviridae; Signal Transduction; Smad6 Protein; Smad7 Protein; Trans-Activators; Transduction, Genetic; Transforming Growth Factor beta; Up-Regulation

The aim of the present study was to determine the relationships between bone morphogenetic proteins (BMPs), BMP receptor type IA and carcinogenesis of oral epithelium. A retrospective study was performed on material obtained from oral mucosa, including nine cases of normal mucosa (NB), eight cases of nonspecific chronic inflammation (NCI), seven cases of hyperkeratosis (HK), five cases of squamous cell papilloma (SCP), 29 cases of squamous cell carcinoma (SCC) with various grades of differentiation and 10 cases of epithelium adjacent to carcinoma (EAC). Six cases of NB from hard palate (NHP) were chosen as a control group. The benign groups consisted of NCI, HK and SCP. The antibodies against BMP-2/4, -5, receptor BMPR-IA and purified bovine BMP (bBMP-McAb) were utilised using an immunocytochemical method. The results demonstrated that the immunostaining of BMP-2/4, BMP-5, BMPR-IA and bBMP-McAb was weak and not consistent in normal and benign groups. The immunoreactivity level was independent of the clinical and pathological grading of SCC. All cases of SCC showed positive staining for BMP-2/4, BMP-5, BMPR-IA and bBMP-McAb except for three cases and one case of SCC which negatively stained for BMP-2/4 and BMP-5, respectively. The staining intensity and proportion of the positively stained cells were markedly increased in SCC when compared with that of the normal and benign groups except for EAC. The metastatic carcinoma cells in lymph nodes were strongly and positively stained for BMP-2/4 and BMP-5 when compared with the primary lesions. Our results indicate that there was an overexpression of BMP-2/4, BMP-5, bBMP-McAb and BMPR-IA in the high-risk premalignant and malignant lesions of oral epithelium. Our findings suggest that BMP-2/4 and BMP-5 but not BMPR-IA might be involved in the metastasis of oral carcinoma cells.

Topics: Analysis of Variance; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 5; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Proteins; Carcinoma, Squamous Cell; Case-Control Studies; Cell Membrane; Cytoplasm; Epithelium; Humans; Immunohistochemistry; Leukoplakia, Oral; Lymphatic Metastasis; Mouth Mucosa; Mouth Neoplasms; Neoplasm Proteins; Palate; Papilloma; Precancerous Conditions; Protein Serine-Threonine Kinases; Receptors, Growth Factor; Retrospective Studies; Statistics, Nonparametric; Transforming Growth Factor beta

Transforming growth factor beta1 (TGF-beta1) acts as a tumor suppressor at early stages of carcinogenesis, however, it has also been suggested to promote tumor progression at late stages. To determine at which stage and by what mechanisms this functional switch occurs, we have generated gene-switch-TGF-beta1 mice in which TGF-beta1 transgene expression can be induced in skin tumors at specific stages. These mice were exposed to a chemical carcinogenesis protocol, which allows tumorigenesis to develop in progressive stages from benign papillomas to malignant carcinomas. Remarkably, TGF-beta1 transgene induction in papillomas rapidly induced metastasis. This function is in sharp contrast to its tumor suppressive effect when TGF-beta1 transgene expression was induced early in the protocol. Transgenic papillomas exhibited down-regulation of TGF-beta receptors and their signal transducer, the Smads, and loss of the invasion suppressor E-cadherin/catenin complex in the cell membrane. These molecules were lost only in malignant carcinomas in control mice at a much later stage. Furthermore, transgenic papillomas exhibited elevated expression of matrix metalloproteinases and increased angiogenesis. Our study suggests that TGF-beta1 overexpression may directly induce tumor metastasis by initiating events necessary for invasion. Down-regulation of TGF-beta signaling components in tumor epithelia selectively abolishes growth inhibition, thus, switching the role of TGF-beta1 to a metastasis promoter.

Topics: Animals; beta Catenin; Cadherins; Cytoskeletal Proteins; Desmoplakins; Down-Regulation; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Inbred ICR; Mice, Transgenic; Neoplasm Metastasis; Neovascularization, Pathologic; Papilloma; Signal Transduction; Skin Neoplasms; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transgenes

The symposium on Virus Infections and Tumors of the Oral Mucosae was organized as a joint meeting of the Arbeitskreis Oralpathologie and Oralmedizin and the Arbeitsgemeinschaft Dermatologische Infektiologie (ADI) der Deutschen Dermatologischen Gesellschaft. The main topics of the meeting were herpes virus infections, human papillomavirus (HPV) infections and human immunodeficiency virus (HIV) infections of the oral mucosae. Clinically both diagnostic, differential diagnostic and therapeutic aspects of the virus-associated diseases were discussed in several presentations. Another important issue was the role of these viruses, particularly of HPVs, in the origin and development of oral cancer. Apparently besides smoking and alcohol other risk factors comprise high risk HPVs, immunodeficiency and possibly also genetic factors. Whether neonatal early infections may predispose children to a specific cancer risk in their future life is still at a level of discussion. Some arguments, however were shown that tonsillar carcinoma, which shows the highest prevalence of the high-risk HPV 16- DNA sequences between all oral cancer, is possibly an epidemiologically and etiologically distinct tumor. It is argued that this tumor is probably less dependent on classical carcinogens than other oral malignant tumors.

Topics: DNA, Viral; Genes, p53; Herpesviridae Infections; HIV Infections; Humans; Leukoplakia, Oral; Lichen Planus, Oral; Mouth Neoplasms; Papilloma; Papillomaviridae; Papillomavirus Infections; Transforming Growth Factor beta; Tumor Virus Infections

Mutations in the transforming growth factor beta type II receptor (TGF-betaRII) have been identified in human cancers, which suggests a causal role for the loss of TGF-betaRII in cancer development. To directly test this in vivo, we have generated transgenic mice expressing a dominant negative TGF-betaRII (delta betaRII) in the epidermis, using a truncated mouse loricrin promoter (ML). ML.delta betaRII transgenic mice exhibited a thickened skin due to epidermal hyperproliferation. When these mice were subjected to a standard two-stage chemical carcinogenesis protocol, they exhibited an increased sensitivity, with an earlier appearance and a 2-fold greater number of papillomas than control mice. In addition, papillomas in control mice regressed after termination of 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment; whereas ML.delta betaRII papillomas progressed to carcinomas. Furthermore, TPA promotion alone induced papilloma formation in ML.delta betaRII mice, which suggests an initiating role for delta betaRII in skin carcinogenesis. ML.delta betaRII tumors also exhibited increased neovascularization and progressed to metastases, although the primary tumors were still classified as carcinoma in situ or well-differentiated carcinomas. Increased expression of vascular endothelial growth factor, an angiogenesis factor, and decreased expression of thrombospondin-1, an angiogenesis inhibitor, were also observed in ML.delta betaRII tumors. The increased angiogenesis correlated with elevated endogenous TGF-beta1 in ML.delta betaRII tumors. These data provide in vivo evidence that inactivation of TGF-betaRII accelerates skin carcinogenesis at both earlier and later stages, and increased angiogenesis is one of the important mechanisms of accelerated tumor growth and metastasis.

Topics: Animals; Carcinogens; Epidermis; Genes, ras; Genetic Predisposition to Disease; Humans; Mice; Mice, Transgenic; Neoplasm Metastasis; Neovascularization, Pathologic; Papilloma; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Skin Neoplasms; Transforming Growth Factor beta; Tumor Suppressor Protein p53

The SENCAR stock of mice has proved to be a useful model in dissecting out the multistage nature as well as the critical mechanisms involved in skin tumorigenesis. This outbred stock was selectively bred to be susceptible to initiation with 7,12-dimethylbenz[a]anthracene (DMBA) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). In order to obtain mice more suitable for genetic analyses of tumor susceptibility and tissue transplantation studies, several inbred lines of mice were derived from the SENCAR stock. One of these lines, the SSIN mice, has a higher susceptibility to tumor promotion compared to the SENCAR stock but is very resistant to tumor progression. On the other hand, the SENCAR B/Pt mice, derived also from the outbred stock, not only have a tumor promotion susceptibility almost identical to the SSIN mice, but they also have a high susceptibility to tumor progression. In order to understand the nature of the phenotypic differences between these two inbred lines we have characterized them using several parameters and markers that are associated with the progression of papillomas to squamous cell carcinoma (SCC). In this sense we analysed the tumor multiplicity and SCC incidence, and the expression of markers of progression and cell cycle related proteins in papillomas derived from both strains. Our results showed that while both strains have a similar papilloma multiplicity and incidence the SENCAR B/Pt mice have 67% incidence of SCC, compared to 0% in the SSIN. SENCAR B/Pt papillomas at 30 weeks of promotion have a higher and aberrant expression of K13, and loss of connexin 26. TGF-beta1 was found to be over-expressed in the suprabasal and superficial cells in the SENCAR B/Pt papillomas, while it was only expressed in the superficial cell layer in those derived from SSIN. The SENCAR B/Pt papillomas also showed an enlarged proliferative compartment with overexpression of cyclin D1 and PCNA as seen by immunohistochemistry and Western blot.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Antigens, Surface; Biomarkers, Tumor; Carcinogens; Cyclin D1; Disease Susceptibility; Female; Immunohistochemistry; Integrin alpha6beta4; Integrins; Mice; Mice, Inbred Strains; Papilloma; Proliferating Cell Nuclear Antigen; Skin Neoplasms; Tetradecanoylphorbol Acetate; Time Factors; Transforming Growth Factor beta

Retinoic acid (RA) was topically applied to the skin of Sencar mice during the promotion phase of specific tumor induction protocols that produce papillomas at low (12-O-tetradecanoylphorbol-13-acetate promoted, TPA) or high (mezerein-promoted) risk for premalignant progression and malignant conversion. RA consistently reduced the yield of papillomas and carcinomas in both protocols, but the frequency of malignant conversion in papillomas that emerged during RA treatment was not reduced. When TPA was reapplied after cessation of RA treatment, the number of papillomas increased 2-fold, suggesting that RA had not eliminated initiated cells. In vitro, RA prevented the emergence of transformed keratinocytes in an assay that mimics malignant conversion, suggesting that RA can suppress conversion if applied during the stage of premalignant progression. Examination of tumor markers at weeks 14 and 22 of the tumor-induction experiments in vivo indicated that papillomas evolving during RA treatment exhibited a phenotype of high progression risk, even in the TPA-promoted groups. In the majority of these tumors, the alpha6beta4 integrin and retinoid X receptor alpha transcripts were detected suprabasally, indicating an advanced state of premalignant progression. RA-treated tumors also expressed higher levels of transcripts for transforming growth factor (TGF)-beta1 and localized TGF-beta1 peptide in the basal portions of the tumor fronds. Because up-regulated expression of TGF-beta1 suppresses papilloma formation, these studies suggest a mechanism whereby RA can prevent papilloma eruption via a TGF-beta intermediate, but papillomas resistant to RA may have altered TGF-beta signaling and progress to carcinomas at an increased frequency.

Topics: Administration, Topical; Animals; Anticarcinogenic Agents; Antineoplastic Agents; Biomarkers, Tumor; Carcinogens; Carcinoma, Basal Cell; Cell Transformation, Neoplastic; Disease Progression; Diterpenes; Female; Immunohistochemistry; Mice; Mice, Inbred BALB C; Mice, Inbred SENCAR; Papilloma; Phenotype; Precancerous Conditions; Receptors, Retinoic Acid; Retinoid X Receptors; Risk Factors; Skin Neoplasms; Terpenes; Tetradecanoylphorbol Acetate; Transcription Factors; Transforming Growth Factor beta; Tretinoin

The mRNA expression and autoregulation of expression of the three isoforms of transforming growth factor-beta (TGFbeta) were examined in the mouse skin carcinogenesis model by northern analyses. We found that TGFbeta3 mRNA levels followed a pattern similar to those of TGFbeta1 during carcinogenesis: the levels were somewhat low in normal skin but became highly overexpressed in late-stage papillomas and squamous cell carcinomas (15- to 20-fold higher than in normal skin). On the other hand, the TGFbeta2 mRNA levels remained relatively low in all benign and malignant tumors, even though the levels were higher than the nearly undetectable levels in normal skin. In a squamous cell carcinoma cell line (CH72), stable transfection and expression of a mutated simian TGFbeta1 cDNA producing bioactive TGFbeta1 significantly downregulated (mean greater than ten-fold) TGFbeta2 mRNA levels and modestly downregulated (about twofold) murine TGFbeta1 expression but had no effect on TGFbeta3 mRNA. In contrast, treatment of all CH72 clones with exogenous TGFbeta1, TGFbeta2, or TGFbeta3 either had no effect or slightly downregulated TGFbeta1 mRNA, upregulated TGFbeta2 mRNA expression an average of twofold to threefold, and strongly upregulated (mean 13- to 27-fold) TGFbeta3 mRNA levels. TGFbeta treatment of primary cultures of mouse skin keratinocytes upregulated all three TGFbeta mRNA levels slightly to moderately (1.3- to 5-fold). Thus, although TGFbeta1 and TGFbeta3 mRNA expressions were apparently coordinately upregulated during mouse skin carcinogenesis, the three TGFbeta mRNAs were differentially regulated by stable transfection of active TGFbeta1 versus exogenous TGFbeta treatment in CH72 cells and by TGFbeta treatments of normal keratinocytes versus carcinoma CH72 cells.

Topics: Animals; Blotting, Northern; Carcinoma, Squamous Cell; Gene Expression Regulation, Neoplastic; Mice; Papilloma; RNA, Messenger; Skin Neoplasms; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

We have previously shown that transforming growth factor-beta 1 (TGF beta 1) mRNA is consistently overexpressed in squamous cell carcinomas relative to normal mouse skin. Here we show that 92-kDa type IV collagenase (matrix metalloproteinase) (MMP-9) mRNA was likewise progressively overexpressed during mouse skin carcinogenesis. To determine if overexpression of MMP-9 and TGF beta 1 are linked, we stably transfected a bioactive TGF beta 1 into a mouse skin squamous cell carcinoma cell line (CH72), which resulted in about twofold to three-fold higher levels of secreted active TGF beta 1. Active TGF beta 1-transfected cells grew only slightly, but not significantly, more slowly in vitro and in vivo than vector-only transfectants. Two clones overexpressing active TGF beta 1 secreted much reduced levels of MMP-9 activity, as determined by zymogram analyses. However, treatment of these clones with 40 pM exogenous TGF beta 1 for 48 h enhanced secretion of MMP-9 activity. Constitutive mRNA expression of MMP-9 was reduced twofold to 70-fold in five untreated active TGF beta 1-transfected clones relative to the other transfectants. In contrast, treatment with 40 pM exogenous TGF beta 1 induced MMP-9 mRNA expression in a time-dependent fashion, from twofold to fourfold after 4 h to a maximum of 12- to 19-fold after 24-48 h. Induction of MMP-9 mRNA was dose dependent at TGF beta 1 concentrations of 4-400 pM. Thus, stable transfection of bioactive TGF beta 1 downregulated whereas exogenous TGF beta 1 treatment upregulated MMP-9 activity and expression. Treatment of transfectants with a neutralizing TGF beta 1 antibody slightly downregulated constitutive MMP-9 mRNA (20-30%) but completely blocked induction by exogenous TGF beta 1. Thus, the effect of TGF beta 1 transfection was not due to secreted TGF beta 1 but may have been a secondary effect.

Topics: Animals; Antibodies, Blocking; Blotting, Northern; Carcinoma, Squamous Cell; Collagenases; Dose-Response Relationship, Drug; Female; Matrix Metalloproteinase 9; Mice; Mice, Nude; Mutation; Papilloma; RNA, Messenger; Skin Neoplasms; Time Factors; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

The p21(CIP1/WAF1) protein is considered a downstream effector of tumor suppression by p53. We have previously demonstrated that p53 null keratinocytes have lower basal p21(CIP1/WAF1) mRNA levels and that tumors derived from these cells following transduction with the v-ras(Ha) oncogene grow faster than wildtype keratinocytes and rapidly progress to undifferentiated carcinomas (Cancer Res 54: 5584-5592, 1994). In this study, primary keratinocytes differing in p21(CIP1/WAF1) gene dose were transduced with v-ras(Ha) encoding retrovirus and grafted to nude mouse hosts to test whether the p53 null phenotype is mediated through p21(CIP1/WAF1). Resulting tumors from all genotypes were well differentiated papillomas; focal carcinomas were observed in 43, 30 and 44% of papillomas derived from +/+, +/- and -/- keratinocytes, respectively. p21(CIP1/WAF1) deficient keratinocytes expressing v-ras(Ha) do not display the degree of increased growth observed in p53 deficient tumors in vivo or the decreased responsiveness to negative growth regulation by Ca2+ in vitro. These results suggest that p21(CIP1/WAF1) does not regulate the differentiated phenotype or malignant progression of v-ras(Ha) initiated keratinocytes and that additional functions of the p53 protein other than transcriptional regulation of the p21(CIP1/WAF1) gene are required for p53 mediated tumor suppression.

Topics: Animals; Calcium; Carcinoma; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Fibroblasts; Gene Dosage; Genes, ras; Immunohistochemistry; Keratinocytes; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Papilloma; Retroviridae; Skin Neoplasms; Transcription, Genetic; Transforming Growth Factor beta; Tumor Suppressor Protein p53

TGFbeta1 has been implicated in cell cycle control and carcinogenesis. To address the exact function of TGFbeta1 in skin carcinogenesis in vivo, mice with TGFbeta1 expression targeted to keratinocytes were subjected to long-term chemical carcinogenesis treatment. TGFbeta1 showed biphasic action during multistage skin carcinogenesis, acting early as a tumor suppressor but later enhancing the malignant phenotype. The transgenics were more resistant to induction of benign skin tumors than controls, but the malignant conversion rate was vastly increased. There was also a higher incidence of spindle cell carcinomas, which expressed high levels of endogenous TGFbeta3, suggesting that TGFbeta1 elicits an epithelial-mesenchymal transition in vivo and that TGFbeta3 might be involved in maintenance of the spindle cell phenotype. The action of TGFbeta1 in enhancing malignant progression may mimic its proposed function in modulating epithelial cell plasticity during embryonic development.

Topics: Animals; Carcinoma; Carcinoma, Squamous Cell; Cells, Cultured; Female; Immunologic Techniques; Integrins; Keratins; Mice; Mice, Transgenic; Papilloma; Skin Neoplasms; Transforming Growth Factor beta

Skin papillomas and squamous cell carcinomas (SCCs) are induced in mice by tumor initiation with a carcinogen followed by tumor promotion with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). These usually arise from preneoplastic lesions characterized by epidermal proliferation and hyperplasia, dermal edema, and inflammation. To evaluate the role of polypeptide growth factors in chemically induced skin carcinogenesis, we used transgenic mice carrying the cDNA for a TGF-beta related molecule, bone morphogenetic protein-4 (BMP-4), under the control of the regulatory elements of the cytokeratin IV* gene in a skin carcinogenesis protocol. Control non-transgenic littermates and BMP-4 transgenic mice were treated with a single dose of a carcinogen, N-methyl-N'-nitrosoguanidine (MNNG), and biweekly with the tumor promoter TPA for 9 months. In control littermates TPA induced epidermal hyperproliferation, atypia with "dark" cells, and dermal inflammation, resulting in papillomas and SCCs in 13 of 26 animals tested. In BMP-4 transgenic mice, TPA treatment induced the expression of the BMP-4 transgene in interfollicular epidermis but only minimal epidermal thickening, hyperproliferation, and inflammation were noted after the initial dose of TPA. Furthermore, the mitotic indices in transgenic epidermis after 9 months of TPA treatment were significantly lower than the corresponding indices from untreated transgenic epidermis. Consequently, none of the 22 transgenic animals tested developed papillomas or SCCs. In conclusion, we have shown that the TPA induced expression of the BMP-4 transgene blocks proliferation and inflammation in skin, steps that are critical to the subsequent formation of papillomas and SCCs and we characterized an inducible promotersystem which expresses polypeptides in interfollicular epidermis after exogenous stimulation.

Topics: Animals; Bone Morphogenetic Proteins; Bromodeoxyuridine; Carcinoma, Squamous Cell; Cell Division; Epidermis; Methylnitronitrosoguanidine; Mice; Mice, Transgenic; Papilloma; Proteins; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta

Epidermal keratinocyte cultures were established from newborn mice expressing a null mutation in the p53 gene to explore the contribution of p53 to epidermal growth regulation and neoplasia. Keratinocytes were initiated by transduction with a replication-defective retrovirus encoding the v-rasHa oncogene and grafted onto nude mouse hosts. Tumors arising from keratinocytes heterozygous or null for functional p53 in the presence of v-rasHa have growth rates approximately 5-fold higher than those derived from p53(+/+) controls and rapidly form carcinomas, in contrast to the benign phenotype observed in p53(+/+)/v-rasHa grafts. In vitro, p53-deficient keratinocytes with and without v-rasHa expression display decreased responsiveness to the negative growth regulators transforming growth factors beta 1 and beta 2. In combination with v-rasHa, p53-deficient keratinocytes also exhibit decreased responsiveness to elevated Ca2+. These differences between genotypes cannot be attributed to changes in transforming growth factor beta receptor types present or altered levels of epidermal growth factor receptor and are independent of c-myc transcript levels. mRNA expression for the p-53 inducible protein WAF1 correlates with p53 gene dosage, but low levels are still detectable in p53(-/-) keratinocytes. The altered responsiveness of p53 deficient keratinocytes to negative growth regulators may provide a growth advantage to such cells in vivo and render them more susceptible to genetic alterations and malignant conversion.

Topics: Amino Acid Sequence; Animals; Animals, Newborn; Base Sequence; Calcium; Carcinoma; Cell Division; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; ErbB Receptors; Gene Deletion; Genes, myc; Genes, p53; Genes, ras; Genotype; Keratinocytes; Mice; Molecular Sequence Data; Papilloma; RNA, Messenger; Skin Neoplasms; Transforming Growth Factor beta

Expression of transforming growth factor beta 1 (TGF beta 1) protein was examined in chemically induced benign skin tumors with genetically defined empirical risks for malignant conversion. Benign tumors induced in mice which have both alleles of the p53 gene deleted have a malignant conversion frequency of approximately 50%, whereas similar tumors induced in wildtype and heterozygous p53 mice have conversion probabilities of 3 and 8%, respectively (Kemp et al., Cell, 74: 813-822, 1993). The TGF beta 1 antibody, anti-CC (1-30-1), was shown to stain either the proliferative keratinocyte compartment of the tumor or the tumor stroma, whereas another TGF beta 1 antibody, anti-LC (1-30-1), stained highly differentiated granular cells of the tumors. A strong correlation was found between staining of the proliferative keratinocyte compartment of tumors with the anti-CC (1-30-1) antibody and tumor genotype. Only 18% (6 of 32) of homozygous p53 null tumors showed any basal keratinocyte staining with this antibody, whereas over 80% (32 of 38) of heterozygous and wild-type tumors showed positive staining. Additionally, in most tumors examined, the spatial distribution of staining for the proliferating cell nuclear antigen appeared to be mutually exclusive with that of TGF beta 1 on adjacent serial sections. This suggests that, in these cases, tumor keratinocytes are sensitive to negative growth regulation by TGF beta. TGF beta 1 protein staining in benign tumors is thus prognostic for a low probability of malignant conversion, and its expression may be mechanistically involved in limiting malignant conversion since, at the benign tumor stage examined, keratinocytes are still sensitive to growth inhibition by TGF beta 1.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Gene Deletion; Genes, p53; Immunohistochemistry; Keratinocytes; Mice; Mice, Mutant Strains; Papilloma; Prognosis; Proliferating Cell Nuclear Antigen; Skin Neoplasms; Transforming Growth Factor beta

Mouse skin carcinomas arise from a small subpopulation of benign papillomas with an increased risk of malignant conversion. These papillomas arise with limited stimulation by tumor promoters, appear rapidly, and do not regress, suggesting that they differ in growth properties from the majority of benign tumors. The transforming growth factor beta (TGF-beta) proteins are expressed in the epidermis and are growth inhibitors for mouse keratinocytes in vitro; altered TGF-beta expression could influence the growth properties of high-risk papillomas. Normal epidermis, tumor promoter-treated epidermis, and skin papillomas at low risk for malignant conversion express TGF-beta 1 in the basal cell compartment and TGF-beta 2 in the suprabasal strata. In low-risk tumors, 90% of the proliferating cells are confined to the basal compartment. In contrast, the majority of high-risk papillomas are devoid of both TGF-beta 1 and TGF-beta 2 as soon as they arise; these tumors have up to 40% of the proliferating cells in the suprabasal layers. Squamous cell carcinomas are also devoid of TGF-beta, suggesting that they arise from the TGF-beta-deficient high-risk papillomas. In some high-risk papillomas, TGF-beta 1 loss can occur first and correlates with basal cell hyperproliferation, while TGF-beta 2 loss correlates with suprabasal hyperproliferation. Similarly, TGF-beta 1-null transgenic mice, which express wild-type levels of TGF-beta 2 in epidermis but no TGF-beta 1 in the basal layer, have a hyperproliferative basal cell layer without suprabasal proliferation. In tumors, loss of TGF-beta is controlled at the posttranscriptional level and is associated with expression of keratin 13, a documented marker of malignant progression. These results show that TGF-beta expression and function are compartmentalized in epidermis and epidermal tumors and that loss of TGF-beta is an early, biologically relevant risk factor for malignant progression.

Topics: Animals; Base Sequence; Carcinoma, Squamous Cell; Cell Division; Cell Transformation, Neoplastic; Epidermal Cells; Epidermis; Female; Keratins; Mice; Mice, Transgenic; Molecular Sequence Data; Papilloma; Risk; Skin Neoplasms; Transforming Growth Factor beta

Topical weekly application of 64 micrograms of benzo[a]pyrene (BAP) for 4 wk induced transforming growth factor (TGF)-beta 1 mRNA in the epidermis of Swiss (ICR) mice, with a maximum at 6-12 h after the last treatment. The increase in TGF-beta 1 mRNA concentration was accompanied by an increase in immunohistochemically detectable intracellularly localized TGF-beta 1 protein in the suprabasal epidermis and by the appearance of extracellularly localized TGF-beta 1 in the basal layers. A dose rate of 16 micrograms/wk for 4 wk was unable to induce the same response. In contrast, after 20 weekly topical applications of 16 or 64 micrograms of BAP, an increase in TGF-beta 1 mRNA concentration and the appearance of extracellularly localized protein in the epidermis were observed. These changes in TGF-beta 1 expression were paralleled by changes in epidermal morphology. A similar group of animals treated with 4 micrograms of BAP/wk for 20 wk did not respond differently from untreated controls. Papillomas resulting from treatment with 16 or 64 micrograms of BAP/wk for 28 wk stained for intracellularly localized TGF-beta 1 predominantly in the differentiating and nondividing layers. Papillomas stained for extracellularly localized TGF-beta 1 solely in the less differentiated and dividing cells. These results suggest that tumorigenesis by BAP involves the induction of cumulative changes in epidermal TGF-beta 1 mRNA and protein concentrations as well as alterations in skin morphology associated with a tumor-promotion process.

Topics: Administration, Topical; Animals; Benzo(a)pyrene; Dose-Response Relationship, Drug; Epidermis; Female; Gene Expression; Immunohistochemistry; Mice; Mice, Inbred ICR; Neoplasm Proteins; Papilloma; RNA, Messenger; Skin; Skin Neoplasms; Skin Physiological Phenomena; Time Factors; Transforming Growth Factor beta

Transforming growth factor beta (TGF-beta) inhibits proliferation of normal keratinocytes, and this response is retained, to variable extents, in benign tumors of the skin (S. Haddow, D. J. Fowlis, K. Parkinson, R. J. Akhurst, and A. Balmain, Oncogene, 6: 1465-1470, 1991). To investigate the profile of TGF-beta biosynthesis during various stages of chemical carcinogenesis of the skin, we used a combination of ribonuclease protection assay, in situ hybridization with gene-specific probes for TGF-beta 1, -beta 2, and -beta 3, and immunohistochemistry with isoform-specific antibodies against TGF-beta 1. Following 12-O-tetradecanoylphorbol-13-acetate treatment of adult mouse skin, there was a rapid induction of TGF-beta 1 protein. Intracellular TGF-beta 1 protein was localized to suprabasal keratinocytes, and the extracellular form was localized predominantly to the dermis. Despite ubiquitous induction of TGF-beta 1 protein by 12-O-tetradecanoylphorbol-13-acetate in various mouse strains, we noted strain-specific differences in the quantitative induction of TGF-beta 1 RNA. Papillomas and carcinomas induced in vivo had elevated levels of TGF-beta 1 RNA within the basal keratinocyte compartment but did not contain significant levels of TGF-beta 1 protein within the tumor. We postulate that the tumor evades TGF-beta 1-controlled negative growth regulation by altered translational and/or posttranslational processing mechanisms of this growth factor. Levels of TGF-beta 2 and -beta 3 RNA were not elevated at any stage of chemical carcinogenesis of the skin.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinoma; Cell Line; Gene Expression Regulation, Neoplastic; Immunohistochemistry; Keratinocytes; Mice; Papilloma; Phorbol Esters; RNA Processing, Post-Transcriptional; RNA, Messenger; Skin Neoplasms; Transforming Growth Factor beta

The influence of an oncogenic v-Ha-ras gene on the expression of TGF-beta and TGF-alpha by mouse keratinocytes and derived tumors has been investigated. Normal mouse keratinocytes cultured as basal cells in 0.05 mM Ca2+ secreted low levels of TGF-beta 2 peptide, and this increased markedly following culture in 1.4 mM Ca2+, retinoic acid, or phorbol esters. In contrast, introduction of a v-Ha-ras gene into normal keratinocytes increased basal expression and secretion of TGF-beta 1 (rather than TGF-beta 2) in response to all three agents. The selective secretion of TGF-beta 1 in v-Ha-ras keratinocytes in response to 1.4 mM Ca2+ occurred even though the four TGF-beta 2 transcripts were induced and the TGF-beta 1 transcript decreased, suggesting that the activated v-Ha-ras gene product regulates expression of the TGF-beta isoforms at the posttranscriptional level. Immunohistochemical analysis of papillomas formed following skin grafting of v-Ha-ras keratinocytes onto nude mice indicated that TGF-beta 1 was abundant in the basal and spinous layers, while there was no expression of TGF-beta 1 in normal skin. In contrast, both normal and neoplastic tissues expressed TGF-beta 2 and TGF-beta 3 in the granular layers. Furthermore, TGF-alpha mRNA expression was also elevated fivefold in cultured v-Ha-ras keratinocytes, and TGF-alpha protein was overexpressed in the grafted papillomas, but there was no detectable expression in normal skin. Elevated expression of both TGF-beta 1 and TGF-alpha in the basal and spinous layers of benign tumors may be important for the high proliferation rate in these tumors as well as for increased proliferation in the suprabasal layer.

Topics: Animals; Cell Differentiation; Down-Regulation; Gene Expression Regulation, Neoplastic; Genes, ras; Keratinocytes; Mice; Mice, Inbred BALB C; Mice, Nude; Papilloma; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured