Anthony J. Dicker

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.

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 (IFNγ) 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 cdk1 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. J. Cell. Physiol. 177:474–482, 1998.

Histone deacetylase inhibitors: novel anticancer agents.

Previous studies have established that the regulation of gene expression is dependent upon the nucleosomal integrity of nuclear DNA. To a large extent, this integrity is dictated by the acetylation status of the core histone particles. The acetylation of histones is, in turn, controlled by the combined activity of specific acetylases and deacetylases. Moreover, disruption of histone acetylases and deacetylases has been linked to a wide variety of human cancers. For this reason, the recent availability of potent and specific histone deacetylase inhibitors has provoked a great deal of interest amongst cancer biologists, oncologists and pharmacologists. Within the past 2- 3 years, several novel histone deacetylase inhibitors have been reported, many of which have already been tested in vivo in mouse models of cancer. In this review we focus on the rationale behind the use of histone deacetylase inhibitors as anticancer agents. Moreover, we review some of the recent findings pertaining to the use of these compounds as anticancer agents.

Amelanotic lentigo maligna and amelanotic lentigo maligna melanoma: a report of three cases mimicking intraepidermal squamous carcinoma

The clinical diagnosis of amelanotic melanoma may pose diagnostic difficulties. We report three cases of amelanotic lentigo maligna, two of which developed an invasive component (lentigo maligna melanoma). The clinical appearances in each case mimicked intraepidermal squamous carcinoma.

Infiltrating basal cell carcinoma: a stellate peri-tumor dermatoscopy pattern as a clue to diagnosis.

Background: Infiltrating basal cell carcinoma (BCC) has associated features that may be readily identified using dermatoscopy. Objective: Investigate a stellate dermatoscopy pattern extending from the peripheral margin of infiltrating BCC. Methods: A total of 741 consecutive cases of BCC were assessed retrospectively using non-polarized dermatoscopy. Following histopathologic examination, cases were categorized into six different BCC subtypes. Infiltrating cases numbered 107. This stellate feature was defined as a geometric star shaped pattern extending outwards from the circumferential peripheral edge of the tumor, and identified by white lines, vessels or uneven skin surface morphology. The percentages of infiltrating subtype within the tumor mass and tumor depth were compared, with and without the stellate pattern. Results: Infiltrating BCC displayed the stellate pattern more than other BCC subtypes. Concordance between the two observers was almost perfect for white lines: Kappa coefficient of 0.87 (95% CI: 0.0.79–0.95) P<0.01 and substantial for vessels: Kappa coefficient of 0.71 95% CI: 0.59–0.84) P<0.01. Folds were only recorded in infiltrating cases (n=3). Compared to other BCC subtypes the stellate pattern had a sensitivity of 31.7% and specificity of 94.1%. A higher mean fraction of the tumor mass containing infiltrating subtype was found when comparing stellate pattern observed to stellate pattern not observed (P<0.01). No statistically significant association was found between the tumor depth with and without the stellate pattern. Conclusion: This study found a higher incidence of the stellate pattern within infiltrating BCC compared to the other BCC subtypes. As the percentage of the infiltrating subtype within the tumors increased the incidence of the stellate pattern also increased.