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Applied Immunohistochemistry & Molecular Morphology:

Expression of E2F-4 Gene in Colorectal Adenocarcinoma and Corresponding Covering Mucosa: An Immunohistochemistry, Image Analysis, and Immunoblot Study

Mady, Hussam H. M.D.*‡; Hasso, Sean B.S.†; Melhem, Mona F. M.D.*‡

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E2F-4 is a transcription factor involved in the transition of the cell from the resting state (G0/G1) to the proliferative stage (S). It has been associated with the p107 and p130 members of the Rb-family and it is responsible for many important growth suppressive functions. E2F-1, one member of the E2F family, has a similar structure to E2F-4; however, both have different mechanisms of action in regulating cell-cycle progression. Although E2F-4 acts mainly as a repressor in the early part of the cell cycle, E2F-1 has the ability to function as both an oncogene and a tumor suppressor gene. In an attempt to identify the role of E2F-4 as a potential mediator of cell proliferation, differentiation, tumorigenesis, and apoptosis in colorectal mucosa comparing with that of E2F-1, the authors examine 20 patients with human colon cancer and their corresponding histologically healthy mucosa by using immunohistochemical methods, computerized quantitative image analysis, and immunoblot analysis. Immunohistochemical studies were performed with formalin-fixed, paraffin-embedded sections stained with a monoclonal antibody against the E2F-4 protein. Apoptosis levels were determined by in situ assay. Positivity was scored by a Computerized Image Analyzer to detect the relative amount of the protein. Immunoblot analysis was performed on protein extracts from snap-frozen tissues of the same specimens. The results show that the expression of E2F-4 was greater in the tumor cells than in their corresponding benign epithelium as determined by immunohistochemical staining and image analysis. This was confirmed by semiquantitative IB analysis of the E2F-4 protein. The labeling index (LI) of E2F-4 in the tumors was inversely proportional to the LI of apoptotic cells. Within these cases, 12 cases showed a very high E2F-4 LI corresponding to low apoptosis LI. Three cases with relatively lower levels of E2F-4 LI were characterized with high apoptotic rates. These data suggest that E2F-4 gene overexpression plays a role in the development of colorectal tumors and appears to play a role in suppressing apoptosis.

Colorectal cancer accounts for more than 90% of the malignant tumors of the large bowel. After lung and breast cancer, it is most common cause of death from malignant disease in Western countries (1). Colorectal tumorigenesis is one of the best-characterized examples of the multistep nature of the cancer. Dominant oncogenes and tumor suppressor genes are consistently involved in colorectal tumorigenesis (2).

The E2F family of transcription factors plays a key role in the control of cell-cycle progression, and some of the members have been implicated in the regulation of apoptotic cell death (3–4). Some family members may act as oncogenes, whereas others act as tumor-suppressor genes (5). The E2F family consists of five members (E2F-1 to E2F-5) that can be divided into two subfamilies based on their structures (6). The E2F proteins have a similar structure, although E2F-1, E2F-2, and E2F-3 are more closely related to each other than are E2F-4 and E2F-5. The E2Fs are both activators and repressors of transcription, and several results suggest that the E2Fs have distinct functions in regulating cell-cycle progression. E2F-4 and E2F-5 act mainly as repressors of E2F-dependent transcription in early parts of the cell cycle; E2F-1, E2F-2, and E2F-3 act as repressors and activators of transcription in later parts of the cell cycle (7). Some in vitro studies found that E2F-1 has both tumor-suppressing and oncogenic activity, and they suggest that E2F-1 overexpression can cause carcinogenesis (7,8).

Studies performed on transgenic E2F-4(−/−) knockout mice models showed delayed intestinal epithelial development with abnormally fewer crypts and proliferating cells than their healthy counterparts (9). E2F-4 is involved in the transition from the resting state (G0) to G1 phase and in facilitating the transactivation of genes necessary for cellular proliferation (10). The tumor-suppressor protein p53 appears to function at the G1 phase of the cell cycle as a checkpoint in response to DNA damage. Mutations in the p53 gene lead to an increased rate of genomic instability and tumorigenesis. E2F-1 has been implicated in increased apoptosis in the absence of p53 (11). No specific correlation between p53 mutation and the E2F-family function is known.

The Rb family of proteins must interact with the E2F family to effect their growth-suppressive functions. E2F-4 has been associated preferentially with p107 and p130 members of the Rb-family (7). This is thought to contribute to the antitumorigenic effect of the Rb family, and disruption of this interaction may lead to the loss of cell growth control.

Some experimental evidence implicating genetic anomalies in the E2F-4 gene in gastrointestinal tumors have been described: the E2F-4 gene contains a polymorphic trinucleotide repeat, coding for a polyserine array, and microsatellite instability affecting this repeat has been documented in gastrointestinal tumors (12,13).

In this study, we examine the expression of two E2F proteins (E2F-1 and E2F-4) and their possible correlation with apoptosis in colorectal cancer by evaluating immunohistochemistry through a computerized image analysis technique that has been previously proven to accurately measure the expression of protein within the cells of interest (14). This was correlated with results from an immunoblot technique by using the same tumors and their corresponding histologically healthy covering epithelium.

© 2002 Lippincott Williams & Wilkins, Inc.


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