Heat Shock Protein in Normal, Premalignant, and Malignant Human Uterine Cervix’

Transformed cells differ from normal cells in their pattern of heat shock protein expression. Here, we report differential expression of a Mr 70,000 heat shock protein (HSP7O) in squamous cell carcinomas of the uterine cervix compared to normal or premabignant uterine cervix. Expression of HSP7O was measured using a mouse mAb against HSP7O by an ELISA. A significant increase in the bevel of expression of HSP7O was observed in fresh tumor cells as compared to normal or dysplastic ones (P < 0.001). The induction of HSP7O in tumor cells subjected to hyperthermia was less than that in normal or premabignant cells. Strong cytoplasmic and nuclear inimunostaining was observed in cancerous tissues using anti-HSP7O mAb, biotinybated rabbit antimouse immunogbobulins, avidin combined in vitro with biotinybated horseradish peroxidase, and diaminobenzidine tetrachboride. HSP7O immunostaining was not detected in normal or dysplastic tissue sections. The results were confirmed by immunoprecipitation using antiHSP7O mAb. These results are the first demonstration of overexpression of HSP7O in squamous cell carcinomas of the uterine cervix as compared to that in dysplastic or normal uterine cervix cells. We conclude that tumor cells differ from normal cells in their pattern of HSP7O expression, and increased levels of HSP7O correlate with an increase in tumor size. INTRODUCTION Stress proteins on HSPs3 are synthesized by eukamyotic and prokamyotic cells under normal conditions, and their synthesis is preferentially increased in cells exposed to physical (heat) or chemical (heavy metals, amino acid analogues, ethanol, ansenite) stress (1). In addition, HSPs are also produced on exposure of cells to inflammatory mediators and cytokines (2), reactive oxygen metabolites (3), and bacteria on viral microorganisms (4, 5). HSPs play an important role in maintenance of cellular homeostasis both under normal conditions and during cellular stress (6). Most HSPs have been grouped into different families based on their size, sequence homologies, and common physiReceived 1/4/95; revised 5/8/95; accepted 5/18/95. I This work was supported by a research grant from the Department of Science and Technology (India). J. K. is a receipient of a Senior Research Fellowship of Council of Scientific and Industrial Research (India). 2 To whom requests for reprints should be addressed. 3 The abbreviation used is: HSP, heat shock protein. obogical functions (7). Among these the members of the highly conserved Mr 70,000 HSP family include the constitutively expressed HSP73 and stress-inducible HSP72. HSP7O family members act as chaperones facilitating protein maturation (8). The members of the HSP7O family have the ability to form complexes with other proteins such as proteins encoded by cellular oncogenes and tumor suppressor genes, thereby altering their functional status (9-1 1). Transformed cells constitutively express higher levels and aberrant HSPs as compared to untransformed cells (12, 13). The bevels of HSPs in cancer cells are higher than in normal cells (11, 14, 15). These observations suggest a role for HSPs in neoplastic diseases. HSPs of the Mr 70,000, 90,000, and 100,000 families have been shown to elicit tumor-specific immunity to tumor challenges in various animal models (16). However little is known about HSP expression in human malignant diseases. Recent reports on comparison of HSP7O expression in human normal and malignant tissues (mainly breast and pancreas) suggest that HSPs play a specific role in the pathogenesis of these carcinomas (17, 18). Cancinoma of the uterine cervix is the second most common cancer in women worldwide after cancer of the breast and the most common in developing countries. The robe of HSPs in pathogenesis of carcinoma of uterine cervix is yet unknown. With the aim of determining whether there is a difference in HSP7O expression in normal and malignant cells and identifying the stage at which alterations in HSP7O expression occurs in the multistep process of development of carcinoma of the uterine cervix, we have studied HSP7O expression in human uterine cervix carcinoma, cervical dysplasia, and normal uterine cervix. We also wished to determine whether the increase in HSP7O expression may be an indicator of the biological stress experienced by malignant cells and may therefore be used to predict disease outcome. MATERIALS AND METHODS Chemicals. MEM and FCS were obtained from GIBCO (Grand Island, NY). Horseradish peroxidase-linked rabbit antimouse immunogbobubins were purchased from Dakopatts (Copenhagen, Denmark). An avidin-biotin kit was purchased from Vector Laboratories, Inc. (Bunbingame, CA). All other reagents were obtained from Sigma Chemical Co. (St. Louis, MO). Ninety-six-well microtiten plates (Linbmo) were obtained from Flow Laboratories (McLean, VA). [35S]methionine was purchased from the Board of Radiation and Isotope Technology (Bombay, India). Tissue Biopsy Specimens. Specimens of normal human uterine cervix, cervical dysplasia, and carcinoma of uterine cervix were obtained from the Out Patients Department of All India Institute of Medical Sciences (New Delhi, India). The specimens were collected in MEM supplemented with 10% FCS. No treatmemt had been given prior to removal of the specimens. Following histopathobogicab confirmation of diagnoResearch. on July 15, 2017. © 1995 American Association for Cancer clincancerres.aacrjournals.org Downloaded from