Endogenous p53 protein generated from wild-type alternatively spliced p53 RNA in mouse epidermal cells.

We previously demonstrated that a wild-type alternatively spliced p53 (p53as) RNA exists in mouse cultured cells and normal mouse tissues at approximately 25 to 33% of the level of the major p53 RNA form. The alternative RNA transcript is 96 nucleotides longer than the major transcript as a result of alternative splicing of intron 10 sequences. The protein expected to be generated from the p53as transcript is 9 amino acids shorter than the major p53 protein and has 17 different amino acids at the carboxyl terminus. We report here that p53as protein exists in nontransformed and malignant epidermal cells and is localized to the nucleus. In addition, p53as protein is preferentially expressed during the G2 phase of the cell cycle and in cells with greater than G2 DNA content compared with the major p53 protein, which is preferentially expressed in G1. The p53as immunoreactivity is elevated and shifted to the G1 phase of the cell cycle following actinomycin D treatment of nontransformed cells but not malignant cells. In view of the dimerization and tetramerization of p53 protein which may be necessary for its DNA binding and transcriptional activation activities, the presence of p53as protein in cells has important implications for understanding the physiological function(s) of the p53 gene.

Functional quantification of DNA-binding proteins p53 and estrogen receptor in cells and tumor tissues by DNA affinity immunoblotting.

Functional assays of proteins can monitor the consequences of defects attributable to posttranslational activating or inhibitory events as well as to genetic mutations. Such assays promise to permit evaluation of cooperating oncogenic or tumor suppressor pathways in cells and tumors. As a step toward realizing this promise, we designed the DNA affinity immunoblotting (DAI) method to measure the activities of multiple sequence-specific DNA-binding proteins simultaneously [initially p53 and estrogen receptor (ER)] in lysates of cells or frozen tumor tissues. DAI is a novel application of biotin/streptavidin affinity chromatography and immunoblotting. The p53 and ER proteins in cell or tissue lysates were bound to biotinylated, specific DNA probes, retrieved using a streptavidin-conjugated matrix, and then quantified in parallel with total protein by immunoblotting. The assay results were reproducible and specifically correlated with the known functional status of p53 in mouse and human cells of known p53 genotype, including those with low levels of p53 protein. ER immunohistochemistry of human breast samples, which is highly correlated with functional status and prognosis in human breast cancer, was also highly correlated with DNA binding activity results by DAI. In contrast, the p53 protein in cells is frequently expressed but inactive, potentially accounting for the lack of strict correlation of p53 immunohistochemical or mutational status with tumor response to chemotherapy. DAI offers a new means of molecular profiling and monitoring of p53 and other DNA-binding protein activities in cells and tumors. DAI has applications in the detection and identification of covalently modified forms of DNA-binding proteins and in the identification of their interacting proteins in complex with DNA.

Altered expression of wild-type p53 tumor suppressor gene during murine epithelial cell transformation.

An epidermal cell model in which initiated, benign tumor-producing and carcinoma stages were derived from a cloned parental cell strain was used to examine p53 expression during multistage epithelial carcinogenesis. Increased steady-state levels of p53 RNA were detected in squamous cell carcinomas compared to papilloma and normal epidermal cells. Nontumorigenic initiated cell precursors of the carcinomas exhibited normal p53 expression, localizing altered p53 regulation to the malignant conversion stage. Immunoprecipitation and Western immunoblot analyses demonstrated elevated levels of p53 protein in the moderately differentiated carcinoma compared to normal cells, and negligible levels of p53 in the poorly differentiated carcinoma cells. Sequence analysis of p53 complementary DNA from normal and carcinoma cells revealed no mutations in the coding or 5'- and 3'-untranslated regions, suggesting a novel mechanism of p53 inactivation.

Tumor progression of murine epidermal cells after treatment in vitro with 12-O-tetradecanoylphorbol-13-acetate or retinoic acid.

Tumor-promoting or antipromoting agents potentially may act directly on initiated squamous epithelial cells or indirectly through effects on normal keratinocytes or immune cells. The purpose of this study was to examine direct effects by comparing in vitro and in vivo treatment of initiated cell populations with 12-O-tetradecanoylphorbol-13-acetate (TPA) or retinoic acid. Keratinocytes were initiated by treatment in vitro with 7,12-dimethylbenz[alpha]anthracene. Replicate cultures of a cloned initiated cell line were exposed to TPA or retinoic acid with acetone as control. After an equivalent number of population doublings, cultured cell sheets were transplanted as skin grafts to athymic nude mice. Replicate grafts from each in vitro treatment group were then treated with TPA or retinoic acid for 8 months. Promotion was quantified by tumor incidence (graft sites with tumor per total sites) and by tumor growth rate. The findings were as follows: (a) TPA increased tumor incidence whether it was applied in vitro or in vivo; (b) TPA in vitro favored more progressive tumors than TPA in vivo; (c) stages of malignant progression from cloned keratinocytes treated in vitro were histologically identical to those following treatment of skin in vivo, including papilloma, dysplastic invasive papilloma, squamous cell carcinoma, and metastasis to lymph node and lung; (d) retinoic acid treatment in vivo reduced tumor incidence and tumor growth rate in initiated cells previously exposed to TPA but not in cells previously exposed to retinoic acid. The results indicated the following: (a) direct effects of TPA on initiated keratinocyte populations were a significant component of tumor promotion; (b) factors in vivo modified the TPA response toward less progressive growth; and (c) the effect of retinoic acid was modulated by prior treatment history.

SPAF, a new AAA-protein specific to early spermatogenesis and malignant conversion.

A novel spermatogenesis associated factor (SPAF) was found to be aberrantly expressed at the malignant conversion stage in a clonal epidermal model of chemical carcinogenesis. Sequence analysis revealed two ATPase modules, classifying this gene as a new member of the AAA-protein family (ATPase associated with diverse activities). Immunohistochemical staining of mouse testis sections with SPAF antibody localized expression to spermatogonia and early spermatocytes in the basal compartment of the seminiferous tubules. Northern and Western analysis of SPAF expression in testes of mice at different developmental stages confirmed its expression at early stages of spermatogenesis. In view of a mitochondrial-localization-like signal, sequence similarities to membrane-associated proteins, ATP binding properties, and intracellular expression patterns in testis, we speculate that SPAF protein may be involved in morphological and functional mitochondrial transformations during spermatogenesis. Ectopic expression of the SPAF gene in malignant epidermal cells may signify adoption of an early germ cell-like phenotype advantageous in malignant conversion.

7,12-Dimethylbenz[a]anthracene-induced mouse keratinocyte malignant transformation independent of Harvey ras activation.

Independent clones of mouse keratinocytes initiated in vitro gave rise to tumor phenotypes typical of mouse skin multistage carcinogenesis and histologically similar to human tumors of the skin, and head and neck. High-molecular-weight genomic DNAs isolated from two 7,12-dimethylbenz[a]anthracene (DMBA)-initiated murine epithelial carcinoma cell lines and one papilloma cell line were examined for transforming activity by transfection into NIH3T3 cells. DNAs from each of these cell lines resulted in the formation of foci morphologically unlike foci containing an activated c-Ha-ras oncogene. Following polymerase chain reaction amplification of the c-Ha-ras gene, Xba I restriction analysis and oligonucleotide differential hybridization did not detect 61st, 12th, or 13th codon mutations. Southern and Northern analysis confirmed that the normal c-Ha-ras gene was not activated by amplification or overexpression. These results provide evidence that 7,12-dimethylbenz[a]anthracene-induced malignant transformation of murine keratinocytes occurred independent of point mutations associated with c-Ha-ras activation. The absence of an activated c-Ha-ras oncogene in these cell lines distinguishes our model from other mouse models of carcinogenesis and may provide a model for functional genetic changes during initiation and progression of human epithelial cancers.

Altered gene expression in a clonal epidermal cell model of carcinogenesis identified by RNA differential display.

We have developed a multistage model system in which a normal mouse keratinocyte clone has been initiated with 7,12-dimethylbenz[a]anthracene and variant clones derived with benign or malignant phenotypes. To identify specific genes altered during mouse skin carcinogenesis, the gene expression patterns of the normal parental epidermal cell, an initiated cell, a benign papilloma, and a poorly differentiated squamous cell carcinoma were compared using RNA differential display. Most alterations in gene expression were observed at malignant conversion, that is, in the poorly differentiated squamous cell carcinoma that is known to have deregulated expression of p53. The sequence of a cloned cDNA fragment lost in the poorly differentiated squamous cell carcinoma was nearly identical to the 3' region of an adhesion-related kinase which is involved in homophilic cell aggregation. It is found in normal epidermal progenitor cells as well as tumorigenic cells with differentiation potential, but not in tumorigenic cells with a poorly differentiated phenotype, suggesting that this adhesion-related kinase may be involved in epidermal cell differentiation. Differential display within the cloned epidermal cell model appears to be useful in detecting and identifying malignant conversion-associated genes which then can be tested directly for their potential role in epithelial carcinogenesis.

Benign and malignant tumor stages in a mouse keratinocyte line treated with 7,12-dimethylbenz[a]anthracene in vitro.

Stable pre-tumorigenic, benign tumor and squamous cell carcinoma stages have been isolated after treatment of a cloned mouse keratinocyte line with 7,12-dimethylbenz[a]anthracene. Intraperitoneal injection and skin grafting of athymic nude mice were suitable for growth of these well-differentiated tumor cells, whereas subcutaneous injection was not.

Alternatively spliced p53 RNA in transformed and normal cells of different tissue types.

The alternatively spliced RNA species of tumor suppressor gene p53, containing an additional 96 bases derived from intron 10, is present at approximately 25 to 30% the level of regularly spliced p53 RNA in both normal epidermal and carcinoma cells. The presence of this alternatively spliced RNA in 10T1/2 fibroblast cells, mouse liver and testis suggests that this alternative splicing may be universal. The level of alternatively spliced p53 RNA was increased coordinately with that of regularly spliced p53 in 10T1/2 cells in response to epidermal growth factor. Immunoprecipitation analysis of epidermal cells using monoclonal antibodies which recognize different epitopes of p53 suggested that distinct p53 proteins may be translated from both RNA species. Considering previous observations on the potential importance of carboxyl terminal sequences in p53 function, knowledge of the ubiquitous presence of alternatively spliced p53 is important for future studies of p53 function in normal cells and in oncogenesis.

Steady-state cultures of human skin.

Pretransplantation cultivation of adult human skin has been optimized for rapid and prolonged outgrowth of epidermal cells from tissue explants using autonomic-perfusion, thin-layer culture technology (steady-state). This system fostered growth of autologous mesenchymal elements via critical control of the culture environment. The resulting cellular outgrowth maintained a balanced epithelial-dermal relationship, contained keratinocytes as well as minority epidermal cells, melanocytes and possibly Langerhans cells. Critical control of culture pH and osmolarity was found to enhance epithelial cell proliferation.

Culture human skin: heterotransplantation.

We have reported that human skin, cultured in a controlled environment system (steady state), yields epidermis-like growth from adult donor split thickness specimens. A procedure for transplantation of such cellular outgrowths to athymic nude mice for functional and morphological evaluations is reported. Successful transplants were definitely scored by the presence of histologic epidermal markers and human glucose phosphate isomerase. We suggest that the transplantation procedures reported here may contribute to the knowledge required for the use of autologous cultured skin in patients with large skin wounds.

Controlled culture of adult human skin: characterization.

Long term growth and differentiation of adult human skin cells has been achieved by more effective control of culture pH, gas phase, osmolarity and nutrient supply at optimized set point values than that possible with conventional batch feed culture. This control was achieved by means of an automatic perfusion, rocker culture apparatus. The following characteristics of these skin cultures particularly lend themselves to application as a source of skin graft material for use in humans requiring large area skin grafts: high culture success rate was achieved in that 12/12 specimens of adult skin exhibited growth to confluence within 4-6 weeks. Cultures demonstrated logarithmic growth for a minimum of 5 weeks, 25-50 fold enhancement in area by 4-6 weeks, 16-fold increase in glucose utilization at 9 weeks, nearly 4-fold increase in total DNA at 5 weeks, and a minimum 6-fold increase in nuclei counts at 5 weeks in culture. The multilayered outgrowth maintained a predominantly epithelial cell composition, but contained normal skin cell types other than keratinocytes, including melanocytes, and dermal fibroblasts, which may be required for long term survival of cultured skin transplanted back to humans. Other than the use of 10% fetal bovine serum in culture medium and denatured bovine collagen substratum, the system did not require heterologous components for plating or growth stimulation. Neither mouse feeder cells, nor the selective pressures of passage were required to obtain growth in surgically useful quantities.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential calcium requirements for growth of mouse skin epithelial and fibroblast cells.

Concentration of extracellular Ca++ optimum for growth of cell types of mesodermal origin have been reported to be up to 100-fold higher than concentrations optimal for epidermal or other epithelial lining cells. In order to examine Ca++ requirements of epithelial v. fibroblastic cells derived from a common tissue source, prior to prolonged culture, freshly isolated mouse epidermal keratinocytes, hair follicle cells and dermal fibroblasts were plated at high density or at clonal density in medium ranging from 0.014 to 1.4 mM Ca++. Epithelial skin cells grew best at Ca++ levels below 0.1 mM while dermal fibroblasts grew best at a Ca++ concentration of 1.4 mM. The epithelial cell types exhibited marked morphologic changes in response to Ca++, while the fibroblasts did not. These results suggest that the variations in Ca++ response between lining epithelium and mesenchymal cells resulted from inherent differences in these cell types, but a mechanism for such differential effects has not yet been defined.

Formation of 8-hydroxydeoxyguanosine within DNA of mouse keratinocytes exposed in culture to UVB and H2O2.

Exposure to UV light contributes to the development of skin cancer. The importance of reactive oxygen species in UV-radiation carcinogenesis has been recognized for some time and several associated DNA base modifications have been identified. In particular, 8-hydroxydeoxyguanosine (8-OHdG) has been well studied as an indicator of oxidative damage to calf thymus DNA exposed to a variety of oxygen-generating systems, including UV light. However, to date, few studies of 8-OHdG have been conducted in cell or animal systems and those in vitro investigations that studied UV exposure have used UVC (< 290 nm), not the UVB (290-320 nm) or UVA (320-400 nm) ranges to which organisms are exposed through sunlight. The objective of this study was to measure 8-OHdG formation in the DNA of cultured mouse keratinocytes exposed to UVB. Using HPLC with electrochemical detection, background levels of 8-OHdG were approximately 6 fmol/micrograms DNA in DNA isolated and digested to the nucleoside level. UVB induced 8-OHdG up to 100% above that for mock-treated cells at a dose of 630 mJ/cm2 (dose-response range: 210-630 mJ/cm2). UVB exposure at 630 mJ/cm2 combined with 5 mM H2O2 elevated 8-OHdG formation up to 280% above that in control cells, whereas H2O2 alone had no effect. These results suggest that factors which increase the generation of reactive oxygen species by UV light may be potent cofactors of UV-radiation carcinogenesis.

Quantitative assay for carcinogen altered differentiation in mouse epidermal cells.

Basal epidermal cells can be selectively maintained as a monolayer in culture medium containing a low ionic calcium concentration of 0.01-0.10 mM. Cessation of proliferation, maturation and shedding of squamous sheets can be induced in this population by increasing the calcium concentration above 0.1 mM. Since alterations in the regulation of proliferation and differentiation are associated with epidermal carcinogenesis in vivo, it appeared reasonable that changes in the phenotypic response to calcium might follow exposure to carcinogens in vitro. Support for this hypothesis was provided by the observation that malignant epidermal cells continued to proliferate when switched from low to high calcium medium, and could thus be selected from a mixture of such cells and a large excess of normal cells which did not survive after induced differentiation. Normal primary epidermal cells were plated in low calcium medium, treated on day 3 with a chemical carcinogen, maintained for 3-9 weeks in low calcium (0.02 mM) and then switched to high calcium medium (1.4 mM). After an additional 4 weeks, surviving epithelial colonies were fixed, stained with rhodamine and counted. Treatment of cultures with 7,12-dimethylbenz[a]anthracene or N-methyl-N'-nitro-N-nitrosoguanidine yielded 4-10 fold more colonies than solvent controls. Colony number was proportional to carcinogen dose for both agents, and increased with time in low calcium prior to selection by calcium increase. Cells obtained from colonies in treated cultures demonstrated characteristic epidermal morphology and keratinization, and could be subcultured, but did not grow in agar or produce tumors in syngeneic hosts. This model system represents a quantitative assay for carcinogen altered epithelial cell differentiation and may select for an early property of preneoplastic epidermal cells.

Differentiation and tumor response to retinobenzoic acid RE-80 in a malignant conversion model.

The synthetic retinobenzoic acid RE-80 was evaluated for its potential as an inductor of tumor cell differentiation and as a chemopreventive agent. A minimally toxic dose of RE-80 in vitro produced morphologic changes typical differentiation in epidermal tumor cell colonies. Indirect immunofluorescence indicated induction of a differentiation-associated keratin of internal stratified epithelia, K13, and inhibition of the differentiation-associated epidermal keratin K1. Cultured cells were skin-grafted to athymic nu/nu mice to evaluate RE-80 effects on early stage malignant progression in vivo. When tumors had grown to 3 to 4 mm in diameter, mice were treated by intraperitoneal injection with RE-80 (67 micrograms, 170 mmol, i.p., two times per week) or vehicle (100 microliters 20% ethanol). Papillomas (benign) and moderately differentiated squamous cell carcinomas were reduced in volume about 4-fold by RE-80 treatment. Larger, poorly differentiated squamous cell carcinomas were unaffected. RE-80 resulted in a lower proportion of proliferative cells (detectable by bromodeoxyuridine incorporation) and a higher proportion of moderately to well differentiated tumors after 40 days of treatment compared with control tumors, which were mainly poorly differentiated squamous cell carcinomas. K13 induction in vitro was correlated with response to retinoid in vivo. Induction of differentiation may be mechanism of the response to RE-80 in vivo since carcinoma cells expressing K13 did not incorporate bromodeoxyuridine and were on a terminal pathway.

Clonal interactions in a human squamous cell carcinoma.

F.2a and B.2, cell clones of the human squamous cell carcinoma SCC-12, were examined to characterize their interactions through the expression of growth factors. F.2a was nontumorigenic yet B.2 was fully tumorigenic when injected into the flanks of athymic nude mice. Combination injections of F.2a and a subtumorigenic level of B.2 produced tumors. F.2a and B.2 overexpressed the 4.8-kb transcript for transforming growth factor-alpha (TGF-alpha) as well as the 10.5- and 5.8- kb transcripts for the epidermal growth factor receptor. Neither clone expressed the transcript for epidermal growth factor, while both expressed transcripts for insulin-like growth factor-I (IGF-I) of 8.15, 4.9, and 1.6 kb and transcripts for its receptor of 8.5 and 6.5 kb. Conditioned medium (CM) from either clone stimulated the growth of itself and the other clone in tissue culture. Both clones produced intracellular TGF-alpha detectable by immunohistochemical staining, but not detectable in CM by enzyme-linked immunosorption assay. IGF-I was detected at low levels in CM by radioimmunoassay. Neutralizing antibodies to TGF-alpha but not IGF-I partially inhibit the growth of both clones in tissue culture. These results suggest that (1) TGF-alpha is active in autocrine signaling (2) IGF-I is not directly stimulatory, and (3) additional factors, as yet unidentified, are present in CM and enhance tumor growth. It is concluded that human squamous cell carcinoma SCC-12 is composed of tumorigenic and nontumorigenic clones which interact to enhance growth.