Indole-3-carbinol - induced growth inhibition can be converted to a cytotoxic response in the presence of TPA+Ca(2+) in squamous cell carcinoma cell lines.

We examined the possibility that I3C, when combined with a differentiation stimulus (TPA+CaCl(2)), would sensitise SCC cells to a differentiation stimulus. We report that I3C induces a profound growth inhibition in SCC cells that is dissimilar to the growth inhibition required to initiate differentiation. Moreover, we report that I3C, when combined with TPA+CaCl(2) treatment, induces a loss of colony forming ability that was differentiation and senescence - independent but was due to delayed cytotoxicity. This study shows that I3C in combination with a PKC activator+Ca(2+) may be a useful therapeutic strategy for treating oral SCC.

Histone hyperacetylation induced by histone deacetylase inhibitors is not sufficient to cause growth inhibition in human dermal fibroblasts.

Use of specific histone deacetylase inhibitors has revealed critical roles for the histone deacetylases (HDAC) in controlling proliferation. Although many studies have correlated the function of HDAC inhibitors with the hyperacetylation of histones, few studies have specifically addressed whether the accumulation of acetylated histones, caused by HDAC inhibitor treatment, is responsible for growth inhibition. In the present study we show that HDAC inhibitors cause growth inhibition in normal and transformed keratinocytes but not in normal dermal fibroblasts. This was despite the observation that the HDAC inhibitor, suberic bishydroxamate (SBHA), caused a kinetically similar accumulation of hyperacetylated histones. This cell type-specific response to SBHA was not due to the inactivation of SBHA by fibroblasts, nor was it due to differences in the expression of specific HDAC family members. Remarkably, overexpression of HDACs 1, 4, and 6 in normal human fibroblasts resulted in cells that could be growth-inhibited by SBHA. These data suggest that, although histone acetylation is a major target for HDAC inhibitors, the accumulation of hyperacetylated histones is not sufficient to cause growth inhibition in all cell types. This suggests that growth inhibition, caused by HDAC inhibitors, may be the culmination of histone hyperacetylation acting in concert with other growth regulatory pathways.

Suppression of keratinocyte growth and differentiation by transforming growth factor beta1 involves multiple signaling pathways.

Transforming growth factor beta1 treatment of keratinocytes results in a suppression of differentiation, an induction of extracellular matrix production, and a suppression of growth. In this study we utilized markers specific for each of these functions to explore the signaling pathways involved in mediating these transforming-growth-factor-beta1-induced activities. In the first instance, we found that the induction of extracellular matrix production (characterized by 3TP-Lux reporter activity) was induced in both keratinocytes and a keratinocyte-derived carcinoma cell line, SCC25, in a dose-dependent manner. Furthermore, transforming growth factor beta1 also suppressed the differentiation-specific marker gene, transglutaminase type 1, in both keratinocytes and SCC25 cells. In contrast, transforming growth factor beta1 inhibited proliferation of keratinocytes but did not cause growth inhibition in the SCC25 cells. Transforming-growth-factor-beta1-induced growth inhibition of keratinocytes was characterized by decreases in DNA synthesis, accumulation of hypophosphorylated Rb, and the inhibition of the E2F:Rb-responsive promoter, cdc2, and an induction of the p21 promoter. When the negative regulator of transforming growth factor beta1 signaling, SMAD7, was overexpressed in keratinocytes it could prevent transforming-growth-factor-beta1-induced activation of the 3TP-Lux and the p21 promoter. SMAD7 could also prevent the suppression of the transglutaminase type 1 by transforming growth factor beta1 but it could not inhibit the repression of the cdc2 promoter. These data indicate that the induction of 3TP-Lux and p21 and the suppression of transglutaminase type 1 are mediated by a different proximate signaling pathway to that regulating the suppression of the cdc2 gene. Combined, these data indicate that the regulation of transforming growth factor beta1 actions are complex and involve multiple signaling pathways.

Functional characterization of cultured cells derived from an intraepidermal carcinoma of the skin (IEC-1).

We have successfully isolated a cell line (IEC-1) from an intraepidermal carcinoma of the skin of a patient and compared its behavior, in vitro, to normal human epidermal keratinocytes (HEK) and squamous cell carcinoma cell lines (SCCs). HEK differentiation comprises an initial growth arrest followed by an induction of squamous differentiation-specific genes such as transglutaminase type 1 (TG-1). Using thymidine uptake and TG-1 induction as markers of proliferation and differentiation, respectively, we were able to show that HEKs and the IEC-1 cells undergo growth arrest and induce TG-1 mRNA expression in response to various differentiation-inducing stimuli, while neoplastic SCC cell lines did not. However, differentiation in HEKs was an irreversible process whereas differentiation of the IEC-1 cells was reversible. Furthermore, growth of IEC-1 cells in organotypic raft cultures revealed differences in their ability to complete a squamous differentiation program compared with that of normal HEKs. The IEC-1 cells also exhibited a transitional phenotype with respect to replicative lifespan; HEKs had a lifespan of 4-6 passages, IEC-1 cells of 15-17 passages, and SCC cells were immortal. These alterations in IEC-1 cell behavior were not associated with functional inactivation or mutations of the p53 gene. These data indicate that the IEC-1 cells, derived from a preneoplastic skin tumor, exhibit differences in their ability to undergo terminal differentiation and have an extended replicative lifespan.

E2F-1 induces proliferation-specific genes and suppresses squamous differentiation-specific genes in human epidermal keratinocytes.

Squamous differentiation of keratinocytes is associated with decreases in E2F-1 mRNA expression and E2F activity, and these processes are disrupted in squamous cell carcinoma cell lines. We now show that E2F-1 mRNA expression is increased in primary squamous cell carcinomas of the skin relative to normal epidermis. To explore the relationship between E2F-1 and squamous differentiation further, we examined the effect of altering E2F activity in primary human keratinocytes induced to differentiate. Promoter activity for the proliferation-associated genes, cdc2 and keratin 14, are inhibited during squamous differentiation. This inhibition can be inhibited by overexpression of E2F-1 in keratinocytes. Overexpression of E2F-1 also suppressed the expression of differentiation markers (transglutaminase type 1 and keratin 10) in differentiated keratinocytes. Blocking E2F activity by transfecting proliferating keratinocytes with dominant negative E2F-1 constructs inhibited the expression of cdc2 and E2F-1, but did not induce differentiation. Furthermore, expression of the dominant negative construct in epithelial carcinoma cell lines and normal keratinocytes decreased expression from the cdc2 promoter. These data indicate that E2F-1 promotes keratinocyte proliferation-specific marker genes and suppresses squamous differentiation-specific marker genes. Moreover, these data indicate that targeted disruption of E2F-1 activity may have therapeutic potential for the treatment of squamous carcinomas. Oncogene (2000).

Cytochrome P450, CYP26AI, is expressed at low levels in human epidermal keratinocytes and is not retinoic acid-inducible.

Retinoids, and their synthetic analogues, are well-established regulators of the squamous differentiation programme both in vivo and in vitro. Despite this, very few studies have focused on the mechanism by which retinoid action is terminated, e.g. metabolism. Recently, a new cytochrome P450 family member (CYP26AI) was cloned. CYP26AI was reported to have substrate specificity for retinoids and to be retinoid-inducible. In this study, we have examined the expression and retinoic acid (RA) inducibility of CYP26AI in human epidermis and cultured keratinocytes. We found very low levels of CYP26AI mRNA expression in both epidermis and keratinocytes. Furthermore, we found no evidence for RA inducibility of CYP26 mRNA expression. This lack of RA inducibility was not due to inactivity of the retinoids, as we show that transglutaminase was still repressed by RA in the same cultures. Despite the low levels of CYP26AI expression in the keratinocytes, the keratinocytes were still capable of significant RA metabolism. In conclusion, our study reports, for the first time, that CYP26AI is unlikely to contribute to RA metabolism in keratinocytes. These studies also indicate that as yet unknown isoforms of cytochrome P450 may be involved in RA metabolism in keratinocytes.

Keratinocyte growth arrest is associated with activation of a transcriptional repressor element in the human cdk1 promoter.

In this study we examined the regulation of cdk1 expression in normal human epidermal keratinocytes (HEKs) and neoplastic keratinocytes. Keratinocytes were growth-arrested by allowing the cells to grow to confluence or by treating them with interferon-gamma (IFNgamma) or 12-O-tetradecanoyl phorbol-13-acetate (TPA). RT-PCR and Western blot analysis demonstrated that cdk1 was profoundly reduced in growth-arrested HEKs when compared with dividing HEKs. In contrast, a squamous carcinoma cell line, SCC25, did not growth-arrest in response to growth inhibitors and did not downregulate cdk1 expression. Transfection of HEKs with a reporter gene driven off a 2.5-kb fragment of the human cdk1 promoter indicated that the downregulation of cdkl upon growth arrest was transcriptional. Deletion mapping of the cdk1 promoter indicated that a repressor region was located between -949 - -722 bp. This repressor region was not operative in the SCC25 cells. Examination of DNA:protein binding complexes by gel-shift analysis indicated that nuclear factors from both proliferative and growth-arrested cells bound to the DNA fragment spanning -949- -722 bp. Further analysis revealed that this binding could be resolved into a constitutive and growth arrest-specific complex that bound in a similar fashion to regions spanning -892 - -831 bp and -831 - -774 bp, respectively. The putative growth arrest-specific complex was not found in contact-inhibited fibroblasts and was found at very low levels in SCC25 cells, indicating that the putative repressor binding was growth arrest-specific and possibly keratinocyte-specific. The binding complexes bound to these two fragments were localized, by competition analysis, to regions -874 - -853 bp and -830 - -800 bp. This is the first report of a transcriptional repressor being operative during keratinocyte growth arrest.

E2F1 messenger RNA is destabilized in response to a growth inhibitor in normal human keratinocytes but not in a squamous carcinoma cell line.

Keratinocyte growth arrest is characterized by a reduction in the activity and expression of E2F1. Here, we examine the role posttranscriptional processing plays in the down-regulation of E2F1 during keratinocyte growth arrest. E2F1 mRNA levels were undetectable within 8 h of exposure to the protein kinase C activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Assays of transcript stability indicated that, in untreated keratinocytes, the t 1/2 of E2F1 mRNA was 6.1 h and, in TPA-treated cells, it was 1.7 h. This destabilization was protein synthesis-dependent. In contrast, a growth inhibitor-resistant carcinoma cell line, SCC25, had a very stable E2F1 half-life that was only moderately reduced following TPA treatment. These data demonstrate that the initiation of keratinocyte growth arrest is associated with a rapid destabilization of E2F1 mRNA. These data are consistent with the proposition that inactivation of the posttranscriptional processing of important growth regulatory genes (e.g., E2F1) may contribute to neoplasia.

E2F as a regulator of keratinocyte proliferation: implications for skin tumor development.

E2F and DP family members are established regulators of the cell cycle. In this study, we examined their activity/expression during keratinocyte growth arrest. Treating human epidermal keratinocytes with the growth inhibitors TPA or IFN-gamma or allowing the cells to reach confluence resulted in 90% inhibition of DNA synthesis, whereas a keratinocyte-derived squamous carcinoma cell line (SCC25) was resistant to growth inhibitors. Gel shift analysis of keratinocytes using an E2F response element indicated that growth arrest was associated with a decrease in all E2F binding complexes. This indicates that growth inhibition is not due to negative regulation by pocket proteins. Conversely, gel shift analysis of growth inhibitor-resistant SCC25 cells showed no decrease in E2F binding. If deregulated E2F expression/activity is involved in tumor development, then the deliberate deregulation of E2F activity may make keratinocytes resistant to growth inhibitors in much the same way as the SCC cells. The HPV16 E7 protein is known to activate E2F. Retroviral infection of keratinocytes with E7-expressing constructs resulted in growth inhibitor resistance, whereas infection with E6 constructs did not. E2F is a heterodimeric complex consisting of E2F family members (1-5) and DP proteins (1-3). Examination of the expression levels for E2F genes and other genes associated with the cell cycle indicated that E2F1 was profoundly decreased in growth-arrested keratinocytes (90%), whereas E2F3, E2F5, and DP1 were not. E2F2 and E2F4 were increased in IFN-gamma-treated keratinocytes but not in TPA-treated or confluent keratinocytes. In contrast, SCC25 cells did not undergo growth arrest and did not downregulate E2F1 mRNA expression in response to growth inhibitors. Our results indicate that E2F DNA binding and in particular E2F1 mRNA expression are associated with keratinocyte proliferation. Our results with the SCC25 cells and the E7-infected cells are consistent with the proposition that deregulated E2F expression/activity (in particular E2F1) may be involved in the unregulated proliferation of skin tumor cells.