Original article
Clinical
p16 Gene Expression in Basal Cell Carcinoma

https://doi.org/10.1016/j.arcmed.2008.06.003Get rights and content

Background

Basal cell carcinoma (BCC) develops predominantly in sun-exposed skin in fair-skinned individuals prone to sunburn. BCC typically occurs in adults. High exposure to ultraviolet (UV) radiation increases rate of developing BCC, a slowly growing tumor that occurs in hair-growing squamous epithelium and rarely metastasizes. In genetic studies, BCC patients have cell-cycle abnormalities of different parts of the signaling pathway. Retinoblastoma regulatory pathway is important in cell cycle arrest. In this pathway, p16INK4a, an inhibitor of Rb pathway, binds to CDK4 and CDK6 competitively with cyclin D1 to prevent phosphorylation of tumor suppressor pRB gene. Alteration of this pathway contributes to development of human cancers and also is effective in skin cancers. In this study, we analyzed mRNA expression using in situ RT-PCR and the role of immunohistochemical expression of p16INK4a in BCC.

Methods

Expression of p16 in ten samples of Iranian paraffin-embedded skin BCC were studied using in situ RT-PCR and immunohistochemistry on p16INK4a gene.

Results

Nuclear and cytoplasmic staining intensity of samples within tumor cells and normal skin tissue illustrates different mRNA and protein expression of p16 gene. mRNA of p16 gene and the expressed protein induce cell cycle proliferation and involve both tumor tissue as well as normal skin tissue. However, in this study it was found that there is significant protein and mRNA expression in BCC cells when compared to normal skin tissue (p <0.05).

Conclusions

p16 gene is involved in the pathogenesis of human skin BCC in view of increased p16 mRNA and expressed protein within tumor cells.

Introduction

Abnormalities of the cell cycle related to molecular overexpression of cyclins/cdks and loss of tumor suppressor functions can cause various malignancies (1). Progression through the cell cycle is controlled by cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitory proteins (2). Mutation of proteins important at different levels of the cell cycle alters the regulation of the cell cycle and then high proliferation of the cells induces cancer. In cancer, mutations have been observed in genes encoding CDK, cyclins, CDK activating enzymes, CKI, CDK substrates, and checkpoint proteins (3).

p16/Rb/E2F regulatory pathway is involved in cell cycle arrest and inactivated in most human cancers. In this pathway, p16INK4a is linked with CDK4 and CDK6 in competition with cyclin D1 and then prevents phosphorylation of tumor suppressor protein Rb, which contributes to form pRb–E2F growth-inhibitory complex 4, 5, 6. p16 as a part of this pathway is a low molecular weight protein, which plays an important role in the pathogenesis of cancers. It is a major CDK inhibitor and tumor suppressor gene (TSG) (7). This gene is one of the most frequently altered genes observed in various human neoplasms. It is believed to encode a negative regulatory protein that prevents cell cycle progression from G1 to S phase by inhibiting the activity role of CDK4 or CDK6/cyclin D complex and subsequently Rb protein phosphorylation (8). Loss of cyclin dependent kinase inhibitor p16INK4a inactivates the Rb family of proteins (9).

Altered p16 gene presents in a high percentage of human tumors and can be inactivated by different mechanisms including deletion, point mutations and hypermethylation. Cells with altered p16 will be uncontrolled so as to proliferate and to proceed through G1 phase. It leads to disruption of the cell cycle and contributes to tumorigenesis of different types of skin cancer (3). BCC is mediated through an upregulation of the CDKI p16INK4a and executed through a functional p16INK4a-cyclin D-Rb pathway. The mechanism of this upregulation of p16INK4a is as yet unknown (10).

Even a skin cancer in the earliest stage will be mediated through a wide range of effects. Expression changes related to the genes indicate their association with the well-known environmental carcinogen of UV radiation, and their effects may be mediated through a wide range of pathways (11). Squamous cell carcinoma (SCC), basal cell carcinoma (BCC), and cutaneous malignant melanoma (CMM) are the three most common types of cancer arising on sun-exposed skin (12). BCC, which is the most common cancer worldwide, is a slowly growing tumor, only occurring in hair-growing squamous epithelium. BCC arises by transformation of basal stem cells located in the hair follicles or basal epidermis. There is no known precursor and it rarely metastasizes. BCCs can develop in both a hereditary and a sporadic form 13, 14. Its incidence increases worldwide by up to 10% per year (15). Several clinical and histological subtypes of BCC may exhibit different patterns of behavior. Aggressive therapy is frequently used for variants including micronodular, infiltrating, or morphea form of BCC (16). The most common form of BCC is nodular (17).

Deregulation of cell cycle control results in uncontrolled cell cycle progression. Alteration of Rb pathway components such as p16 leads to uncontrolled proliferation and results in the formation of cancer such as BCC. p16 is an inhibitor gene in the Rb pathway that can regulate cell cycle progression.

Therefore, in an attempt to elucidate the implication of this gene in cell cycle control, the localization of mRNA and protein expression of p16 in BCC were investigated and compared with the normal skin tissue. In this study, mRNA and protein expression level of p16 gene were detected in BCC tissue using in situ RT-PCR and immunohistochemistry (IHC).

Section snippets

Tissue Section Preparation

Tissues from different parts of the body from ten patients who were diagnosed with BCC were adequately surgically excised and sent for processing to the Shafie Pathology Laboratory, Sirjan, Iran. Tissues were subjected to a standard laboratory processing technique and subsequently stained with hematoxylin and eosin. Tissue sections were reviewed and diagnosed by a consultant pathologist in Iran. Sections were paraffin embedded and further processed for in situ RT-PCR and IHC on the p16 gene.

In Situ RT-PCR

Results

Paraffin-embedded tissue of BCC samples obtained from ten patients revealed increase of p16 mRNA expression in in situ RT-PCR. Protease digestion is a critical step for in situ detection of target DNA or RNA (24). Under the circumstances of this study, 10-min protease exposure gave the best results. DNase exposure of sections prior to in situ RT-PCR is necessary to reduce nuclear DNA and nonspecific amplification of DNA. All slides were tested at least three times for the presence of p16 gene

Discussion

There is increasing knowledge of the molecular basis of tumors and this is being used with increasing frequency to identify underlying molecular changes or presence of specific molecular markers in tumors. Both are contributory to the process of making an accurate diagnosis. Acquisition of this knowledge leads to an appropriate choice of therapy that will determine a favorable outcome for the patient (28). IHC and in situ RT-PCR studies have focused on markers of specific cell and tumor types

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