Anatomic Pathology / UROTHELIAL CELL MALIGNANCY AND PROTEIN KINASE C ISOFORM EXPRESSION Patterns of Protein Kinase C Isoenzyme Expression in Transitional Cell Carcinoma of Bladder Relation to Degree of Malignancy

We determined the pattern of protein kinase C (PKC) isoform expression in human cell lines by Western blotting and immunofluorescent staining techniques. In addition, we examined PKC isoform expression in tissue samples of transitional cell carcinoma (TCC) of the bladder. PKC delta, PKC beta II, and PKC eta were found primarily in the RT4 cell line (low-grade tumor), and PKC zeta was expressed most strongly in the SUP cell line (invasive tumor). In tissue samples of urinary bladder cancer, PKC isoenzymes were expressed differentially as a function of tumor stage and grade; expression of PKC beta II and PKC delta was high in normal tissue and in lowgrade tumors and decreased with increasing stage and grade of TCC. The opposite pattern was seen with PKC zeta. The differences in expression of specific isoenzymes as related to levels of malignancy of the cell lines and tissue samples suggest that the PKC family has an important role in normal and neoplastic urothelium. The protein kinase C (PKC) family of serine-threonine kinases has an important regulatory role in a variety of biologic phenomena.1,2 The family is composed of at least 12 individual isoforms that belong to 3 distinct categories: (1) conventional isoforms (alpha, beta I, beta II, gamma) activated by Ca2+, phorbol esters (12-O-tetradecanoylphorbol13-acetate [TPA]), and diacylglycerol liberated intracellularly by phospholipase C; (2) novel isoforms (delta, epsilon, eta, theta), which also are activated by phorbol esters and diacylglycerol but not by Ca2+; and (3) atypical (zeta, lambda, iota) members of the family, which differ in cystein-rich sequences and are not activated by Ca2+, phorbol esters, or diacylglycerol; PKC mu is considered to be intermediate between the novel and atypical groups. The cellular response to TPA activation can be an increase3,4 or decrease5,6 in the rate of proliferation or no change, depending on the cell type and the particular PKC isoenzymes expressed in different cells. Not all isoenzymes are activated by phorbol esters, and there is heterogeneity in response.7 It is generally considered that the enzymes when quiescent are located in the cytoplasm and on activation are translocated to the plasma membrane.8 Not all isoforms behave in this manner; indeed, not all of them are found in all tissues. Moreover, the pattern of isoform distribution varies among different tissue9 and also may change as a function of phenotype.10 Several studies have reported that the pattern of PKC isoenzyme expression and distribution in a number of cell types may change as cells are transformed from a normal to a proliferative phenotype.10-17 Thus, overexpression of PKC beta I in rat fibroblasts caused these cells to be able to form tumors in nude mice.18 It seems, however, that effects of PKC isoenzyme expression may have different influences depending on the cell type. High expression of PKC alpha Am J Clin Pathol 2001;116:377-385 377 © American Society of Clinical Pathologists Langzam et al / UROTHELIAL CELL MALIGNANCY AND PROTEIN KINASE C ISOFORM EXPRESSION alone in MCF-7 breast cancer cells is associated with a transformed phenotype,15 whereas overexpression of both PKC alpha and PKC beta in these cells resulted in a less aggressive phenotype.16 These reports indicate that the change from the normal to the transformed state may be associated with upregulation or down-regulation of a particular PKC isoform. The profile of isoform expression might aid in the understanding of processes leading to cell transformation and provide information about the grade of malignancy of various types of cancer. Tumors of the urinary bladder of transitional epithelium or urothelium exhibit the entire spectrum of biologic aggressiveness, from low-grade tumors to highly malignant carcinoma.19 This provides an excellent opportunity to examine the possibility that tumorigenicity of transitional cell carcinoma (TCC) may be related to the PKC isoform profile. Until now, the pattern of PKC isoform expression in urinary bladder transitional cells of varying degrees of malignancy has not been determined. The purpose of the present study was to determine the effect of TPA on 3 TCC cell lines of different aggressiveness and to characterize the types of PKC isoenzymes in these cell lines. Materials and Methods Cell Lines of Urinary Bladder Tumors The 3 different human cell lines used in the study were purchased from the American Type Culture Collection (ATCC, Rockville, MD). They were selected on the basis of degree of malignancy as determined by the depositors of the cells at ATCC according to morphologic features and ability to produce tumors in nude mice. The 3 lines were (1) TCCRT4, originating from a TCC, grade I; (2) TCC-T24, originating from a TCC grade II, and (3) TCC-SUP, originating from a TCC, grade III that had metastasized to the bone marrow. Growth of Cells in Culture All cells were plated initially at a density of 105/mL. RT4 and T24 cells were grown in medium containing McCoy 5A solution (90%) and heat-inactivated fetal calf serum (10%), and SUP cells were grown in Eagle MEM with nonessential amino acids (90%) and heat-inactivated fetal calf serum (10%), to which was added a 2-mmol/L concentration of glutamine and a 1-mmol/L concentration of sodium pyruvate. All media contained 0.05 mg/mL of streptomycin and 50 U/mL of penicillin. All cell lines were incubated at 37°C in a 6% carbon dioxide atmosphere. All changes of media during the period of cell growth were done at 37°C. Effects of Phorbol Esters on Cell Morphologic Features TPA was dissolved in dimethyl sulfoxide to give a stock solution of 1 mmol/L. TPA was added to the cultures to a final concentration of 50 nmol/L and was added to culture dishes (90 mm) containing each cell line. The effect of this treatment was examined after 15 and 90 minutes of stimulation. In parallel, the same concentration of dimethyl sulfoxide was added to other dishes to control for possible effects of the diluent.