Ierachmiel Daskal

Cells producing cathepsins D, B, and L in human breast carcinoma and their association with prognosis

Lysosomal proteinases, cathepsins D, B, and L have been associated with malignant tumor progression and with prognosis in various human carcinomas. In the current study, the immunohistochemical localization of cathepsins in tumor cells was correlated with cathepsin protein concentration in breast carcinoma cytosols from 77 patients. Significant correlation was found for cathepsin D (P < .041) and borderline correlation for cathepsin B (P < .055) but not for cathepsin L. We hypothesize that the poor correlation of cysteine cathepsins was attributable to the fact that they were present not only in malignant epithelial cells, but also in infiltrating macrophages and stromal fibroblasts. In addition, tumor-surrounding myoepithelial cells (42% of tumors) and myofibroblasts (26% of tumors) as well as endothelial cells of neovasculature (10% of tumors) all stained specifically for cathepsin B. Two thirds of tumors co-expressed cathepsins B and L in tumor cells, whereas only 17% of tumors co-expressed all 3 cathepsins. Intense immunostaining for cathepsin D of tumor cells was observed in tumors at high TNM stage and tumors having positive lymph nodes. The expression of cathepsin B was independent of established prognostic factors, whereas intense cathepsin L staining in tumor cells was associated with high histological grade. With respect to prognosis of patient survival, only tumor cell-associated cathepsin D (P = .042) and myoepithelial cell-associated cathepsin B (P = .061) showed borderline significance. Cathepsins B and L immunostaining in tumor cells was not prognostic. In contrast, cytosolic levels of cathepsin B correlated with higher rate of relapse. Taken together, these results show the diversity in the cellular distribution of cathepsins in human breast carcinoma, presumably reflecting specific regulation and function of each of the cathepsins during tumor progression.

Cathepsins D, B, and L in transformed human breast epithelial cells

To investigate the regulation of lysosomal enzymes during carcinogenesis, we measured cathepsins (Cats) D, B, and L in MCF-10F, which is a human breast epithelial cell line, and cells evolved after treatment with carcinogen and transfected with c-Ha-ras oncogene. The clones used in this study, MCF-10FTras, D3, D3-1, and D3-1Tras, expressed no estrogen receptors and gradually increased invasive potential, while oncogenetransfected lines were also tumorigenic in SCID mice [16,19]. Cats D, B, and L were determined in the cells and in cell media using enzyme-linked immunosorbent assay (ELISA), specific enzyme activity measurements, and immunocytochemistry. The major intra- and extracellular lysosomal proteinase in these cells was Cat D (30–180 pm/mg), followed by Cat B (2–10 pm/mg) and Cat L (1–5 pm/mg). An inverse relationship between intracellular Cat D levels and invasive potential of carcinogen-treated and c-Ha-ras oncogene-transfected cell lines was observed. No significant changes in extracellular concentration of Cat D precursor in this series of cell lines was observed. Intracellular levels of Cats B and L were unchanged or slightly lower in carcinogentreated D3 and D3-1 cells, as well as in MCF-10FTras. On the other hand, in D3-1Tras cell line, evolving from c-Ha-ras transfected D3-1 line, 3.5 fold and 4.4 fold increases in Cat B and Cat L, respectively, but a 2 fold decrease in Cat D, were observed compared to the parental cell line. Immunocytochemical staining showed a granular, polarized perinuclear and cytoplasmic staining of cathepsins in all cell lines. Cysteine proteinases stained more frequently and more intensely in D3-1Tras compared to other lines, confirming the immunochemical assays. We hypothesize that several molecular events, caused by a carcinogen and an oncogene such as c-Ha-ras, are needed to increase Cat B and Cat L, but not Cat D, expression. Therefore, the cysteine and aspartic lysosomal proteinases are differentially expressed in the breast cell lines with more invasive phenotype.

Racial differences in prostate cancer related to loss of heterozygosity on chromosome 8p12-23.

PURPOSE To determine if there is a racial difference in prostate cancer related to loss of heterozygosity (LOH) on chromosome 8p12-23, the region most frequently altered in prostate cancer. METHODS AND MATERIALS A total of 51 prostate cancer patients, consisting of 23 African Americans and 28 Caucasians, were included in this study. All patients underwent radical prostatectomy, and patients in the two racial subgroups were matched for median serum PSA, Gleason score, and pathological stage of cancer. Paired normal prostate and cancer tissue DNA was isolated and amplified with 13 polymorphic markers mapped to 8p12-23 by radiolabeled polymerase chain reaction. The amplified products were resolved by polyacrylamide gel electrophoresis, autoradiographed, and analyzed for allelic losses. RESULTS The overall incidence of LOH at 8p12-23 was 53%, and 16% showed homozygous deletions. The incidence of LOH in Caucasians was 68% compared to 35% in African Americans. On univariate (p = 0.02) and multivariate logistic regression analysis (p = 0.02), only Caucasian race was a significant predictor for LOH. The other clinicopathologic parameters did not have any significant effect on incidence of LOH. CONCLUSION These results highlight the independent influence of Caucasian race on incidence of LOH at 8p12-23, and suggest that genetic differences at specific tumor suppressor loci may be a factor responsible for racial variations observed in prostate cancer.

Effects of PTX1 Expression on Growth and Tumorigenicity of the Prostate Cancer Cell Line PC-3

PTX1 is a gene identified by subtractive hybridization on the basis that it is expressed in normal prostate and not in prostate carcinoma. It encodes a nuclear protein that is downregulated in prostate carcinoma. Expression constructs containing PTX1 cDNA in both sense and antisense orientations were transfected into prostate tumor cell line, PC-3 cells. The effects of the expression of PTX1 and antisense PTX1 on PC-3 cells were examined using cell growth, proliferation, soft agar, invasion chamber, senescence-associated beta-galactosidase, and nude mice assays. Cells transfected with PTX1 construct in the sense orientation were growth-arrested. These cells displayed multiple morphological changes consistent with cellular senescence, including the expression of a senescence-associated beta-galactosidase. On the other hand, expression of antisense PTX1 RNA in PC-3 cells resulted in uncontrolled cell growth and increase of invasive potential. In nude mice, cells expressing antisense PTX1 grew sixfold faster than the control. These results suggest that PTX1 may play an important role in the growth and tumorigenicity of PC-3 cells.

Flow cytometric determination of ploidy in prostatic adenocarcinoma and its relation to clinical outcome

Flow cytometry was used to measure the DNA content in paraffin-embedded archival specimens of prostatic adenocarcinoma. The specimens were from 49 patients who were found to have adenocarcinoma of the prostate at the time of transurethral resection of the prostate for bladder outlet obstruction. At initial presentation, 34 of these patients had clinically localized disease and 15 had metastatic disease. The authors studied the relationship of DNA ploidy to clinical stage, histologic grade, disease progression, and duration of survival. Their results indicate that regardless of the clinical stage at presentation, the mean time to disease progression is longer in a patient with a DNA diploid tumor when compared with that in a patient with a DNA aneuploid tumor (18.1 months vs 6.5 months, respectively). Additionally, mean time of survival was longer in a patient with a diploid tumor than in a patient with an aneuploid tumor (31.6 months vs 9.6 months, respectively).

End-stage renal disease in sarcoidosis of the kidney

We describe two cases of black women with biopsy-proven sarcoidosis of the kidney who developed end-stage renal disease. Treatment with high-dose glucocorticoids resulted in a good initial response, followed by progressive deterioration of renal function requiring hemodialysis.