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The current study was undertaken to analyse growth and differentiation-related functions of normal keratinocytes (NOK) and an SV40T-immortalized keratinocyte line (SVpgC2a) from buccal mucosa, viewing the latter cell line as a model of a dysplastic epithelium. Morphological and immunohistochemical assessments of organotypic epithelia generated from 10 or 17 d of culture showed three- to five-fold higher apoptotic and proliferative activity in SVpgC2a relative to NOK. Conditions with or without serum (up to 10%) did not significantly influence these parameters in NOK whereas serum supported proliferation of SVpgC2a. Both cell types showed basal expression of collagen IV and laminin 1, indicating basal lamina, as well as vimentin, indicating an activated, proliferative state. Reduced expression of keratin, including the non-keratinizing marker K13, was seen in SVpgC2a. Assessment of proliferative monolayer cultures by microarray showed that NOK transcribed tissue-specific keratins, but also the epidermal keratin K2a, several simple epithelial keratins and low levels of hair keratins. SVpgC2a transcribed keratins seen in epithelial dysplasia, and K2a and hair keratins, albeit at low level. Overall, the results implied aberrant apoptosis, proliferation and keratin expression in the immortalized state of SVpgC2a. Comparison of NOK and SVpgC2a under identical culture conditions may serve to model the progression from a normal to a pre-neoplastic state of buccal epithelium.

Loss of heterozygosity in two in vitro-immortalized human oral keratinocyte cell lines was analysed by polymerase chain reaction using 42 polymorphic microsatellite markers on chromosomes 4, 6, 7 and 15. These chromosomes are regarded as candidates for harbouring genes involved in the immortalization of human cells or tumour-suppressor genes in several tumours, including oral cancers, and karyotypic analysis has revealed that both cell lines have non-random alterations in these chromosomes. No allele losses were detected at any informative loci on chromosomes 4 and 6 in the cell lines, including genomic regions adjacent to putative human tumour-suppressor genes and putative senescence genes. When analysed for loss of heterozygosity on chromosomes 7 and 15, allele losses common to both cell lines were detected in the regions at 7q11.2, 7q21.1-21.3 and 7q31.1. High frequencies of loss of heterozygosity on chromosome 7q in at least two distinct regions, particularly centred around 7q31, are observed in a variety of tumours, including oral squamous-cell carcinoma, suggesting that multiple genes involved in immortalization of these cell lines might be present on chromosome 7q.

Collagenase-3 (matrix metalloproteinase 13; MMP-13) is a novel matrix metalloproteinase, the expression of which to date has only been detected in human breast carcinoma tissue and osteoarthritic cartilage. Here, we show that MMP-13 transcripts are expressed by human HaCaT keratinocytes but not by primary human epidermal keratinocytes. The levels of MMP-13 mRNAs in HaCaT cells were enhanced up to 130- and 45-fold by tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta), respectively. The maximal induction of MMP-13 mRNAs by TNF-alpha was noted after a 6-h incubation, whereas with TGF-beta, the maximal stimulation was observed after 24 h. The up-regulation of MMP-13 mRNA abundance by TNF-alpha and TGF-beta was dependent on protein synthesis and was prevented partially by dexamethasone and retinoic acid. Nuclear run-on assays demonstrated activation of MMP-13 gene transcription by TNF-alpha maximally at the 2-h time point and by TGF-beta after 12 h of treatment. Incubation of HaCaT keratinocytes with TNF-alpha and TGF-beta also increased production of proMMP-13 into the culture media, as detected by Western blotting. Our data indicate that the MMP-13 gene is expressed by transformed epidermal keratinocytes, suggesting a role for MMP-13 in the invasive capacity of human epidermal malignancies.

The ability to form tumors in nude mice developed spontaneously in the human papillomavirus (HPV)-18 immortalized keratinocyte cell line, 18-11, and is shown here to be accompanied by a loss of interleukin 1 (IL-1) alpha and beta expression at both the RNA and protein level. In addition, a separate tumorigenic 18-11 derivative and two cervical carcinoma-derived cell lines, HeLa and Caski, were found to have significantly decreased or lost IL-1 alpha and IL-1 beta expression. Using retroviral expression vectors, we re-established IL-1 expression in tumorigenic 18-11 cells (18-11S3) in an effort to evaluate whether loss of IL-1 expression represented an important phenotypic change in the development of tumorigenicity in these cells. IL-1-expressing 18-11S3 cells showed a range of tumorigenic potential, depending on the type and combination of IL-1 alpha and IL-1 beta expressed. Although 18-11S3 expressing the precursor forms of both IL-1 alpha and IL-1 beta normally found in keratinocytes showed moderate inhibition of tumorigenicity, other IL-1-expressing lines showed complete inhibition of tumor formation. Co-injection of nontumorigenic, IL-1-expressing 18-11S3 with parental 18-11S3 also inhibited tumor formation. These results suggest that maintenance of IL-1 expression may play an important role in preventing progression to tumorigenicity in cervical carcinoma and other epithelial cancers.

The retinoblastoma protein (pRB) and the pRB-related pocket proteins p130 and p107, when bound to DNA via the E2F family of transcription factors, suppress transcription and through this may mediate growth arrest. We show here that in HaCat cells arrested by treatment with TGFbeta the only pocket protein associating with DNA-bound E2F is pRB. This contrasts the situation in HaCat cells arrested via growth factor withdrawal, where we find that both pRB and p130 can bind. The above implies that p130 participates in regulating E2F-dependent genes upon growth factor deprivation but not upon TGFbeta arrest. More importantly perhaps, in TGFbeta-arrested cells pRB alone in association with its partner E2Fs may be in charge and sufficient to control E2F-dependent gene transcription. Although p130 is not associated with a DNA-binding E2F complex in TGFbeta-treated cells, it is present in such cells in its underphosphorylated form. We provide evidence for a serum-induced process that may regulate p130 by a mechanism independent of p130 hyperphosphorylation.

Two human keratinocyte cell lines transformed by human papillomavirus type 16, designated Vp and Up, were compared for their clonal growth potential and clonal origin. Up showed greater anchorage-independent growth in soft agar and higher efficiency of single-cell colony formation than Vp (24.3% compared to approximately 10%). The clonal growth potential of these two cell lines was not related to the level of HPV16 gene expression. Fourteen single cell clones of the Vp and 24 of the Up were selected, propagated and analyzed by Southern and Northern blot analysis. Clonal variations existed among subclones of each cell line and between the two cell lines. These variations included cell morphology, growth potential, and expression levels of involucrin (a differentiation marker of keratinocytes) and of HPV16 mRNAs. The Vp and Up cell lines also showed different patterns of HPV16-DNA integration and RNA transcription. However, all subclones of Vp and subclones of Up displayed identical HPV16 DNA integration and RNA expression patterns. The results suggest that both cell lines were monoclonal in origin and that the host genetic factors play an essential role in determining cell clonality.