J. C. Romijn

Characterization of the prostate-specific antigen gene: a novel human kallikrein-like gene

Using Prostate-specific Antigen cDNA fragments as hybridization probes a clone containing the information for the gene encoding Prostate-specific Antigen was isolated form a human genomic DNA library. The complete gene (about 6 kb) was sequenced and shown to be composed of four introns and five exons. Two major transcription initiation sites were found. The sequence of the promoter region revealed the presence of various well known transcription regulatory elements including a TATA box. A high percentage of homology was found between the Prostate-specific Antigen gene and the hGK-1 gene (82%). This homology extended into the promoter region. Two previously described variant Prostate-specific Antigen cDNAs can now be explained by intron retention and alternative splicing of the primary transcript.

The prostate-specific antigen gene and the human glandular kallikrein-1 gene are tandemly located on chromosome 19.

Using a prostate‐specific antigen cDNA as a hybridization probe, clones containing the kallikrein genes encoding prostate‐specific antigen, human glandular kallikrein‐1 and pancreas/kidney kallikrein were isolated from a human genomic library. Clones containing the prostate‐specific antigen gene and the human glandular kallikrein‐1 gene overlap and span a region of about 36 kb. The two genes are aligned in a head to tail orientation at a mutual distance of 12 kb. Southern blot analysis of DNA from a panel of human‐hamster hybrid cells with specific probes revealed the genes to be situated on chromosome 19. Assuming that the pancreas/kidney kallikrein gene is located in the same cluster, the distance to the prostate‐specific antigen gene and the human glandular kallikrein gene must be at least 15 kb.

Chemosensitivity of prostate cancer cell lines and expression of multidrug resistance-related proteins

The aim of this study was to obtain insight into the role of the multidrug resistance (MDR) phenomenon in hormone-independent progressive prostate cancer. Using immunocytochemistry and Western blotting we determined the expression of P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP), glutathione-S-transferase-pi (GST-pi), Bcl-2, Bax, topoisomerase (Topo) I, II alpha and II beta in the human prostate cancer cell lines PC3, TSU-Pr1, DU145 and LNCaP derivatives LNCaP-R, LNCaP-LNO and LNCaP-FGC. Proliferative activity was assessed by immunocytochemistry. MTT assays were used to determine the sensitivity to etoposide, doxorubicin and vinblastin. Pgp was not expressed in any of the cell lines. MRP was variably expressed. GST-pi was expressed in TSU-Pr1, PC3 and DU145. The expression of Bcl-2 was restricted to TSU-Pr1, whereas Bax was found in all cell lines. Topo II alpha was expressed at the highest level in the rapidly proliferating cell lines TSU-Pr1 and DU145. Topo I and II beta were equally expressed. Resistance profiles varied among the cell lines, with TSU-Pr1 being the most sensitive and LNCaP-LNO relatively resistant. Multiple MDR proteins were expressed in prostate cancer cell lines and may well influence response to chemotherapy. Future functional studies, using chemo-selected MDR models, may further help to determine the mechanism or combination of mechanisms underlying the resistance of prostate cancer to chemotherapy.

Expression of cellular oncogenes in human prostatic carcinoma cell lines

Prostatic cancer is one of the most frequent forms of malignancy in Western countries. Initially, growth of the majority of prostate tumors can be manipulated by endocrine therapy. However, ultimately androgen independent tumors continue to grow. We studied the expression of oncogenes in four different human prostatic carcinoma cell lines: PC 3, PC 133, PC 135, which are androgen independent, and the hormone dependent PC 82 cell line. Large amounts of Ha-ras and myc mRNA were present in all cell lines. Transcripts of fes, int-1 and abl were never detected. In some of the cell lines the presence of N-ras, Ki-ras, myb, fos, fms and sis mRNA was observed. The PC 82 cell line showed, in addition to myc and Ha-ras high levels of fos expression. Inhibition of tumor cell proliferation by withdrawal of androgen was accompanied by a tenfold reduction of the fos mRNA level and a twofold reduction of Ha-ras transcripts. In contrast, the expression of myc was not changed.

Crystal-Cell Interaction Inhibition by Polysaccharides

PURPOSE We studied the effect of polysaccharides on interactions between calcium oxalate monohydrate (COM) crystals and cultured renal cells. MATERIALS AND METHODS Monolayers of Madin-Darby canine kidney (MDCK) cells were incubated with radiolabeled crystals in the presence of various concentrations of natural glycosaminoglycans (GAGs) and semisynthetic polysaccharides (SSPs). RESULTS While most GAGs were found to have relatively little effect, SSPs (SP54, G871 and G872) were potent inhibitors of crystal-cell association. Pretreatment of crystals, but not of cells, was similarly effective, suggesting polysaccharide-induced modification of crystal surface properties. CONCLUSIONS This result further supports the idea that SSPs, and especially G872, are of potential interest for treatment of recurrent stone disease.

Biological effects of hormonal treatment regimens on a transplantable human prostatic tumor line (PC-82).

The effects of hormonal manipulation on the growth of a transplantable human prostatic carcinoma line (PC-82) were studied. The histological pattern of the PC-82 tumor, which still closely resembles the original tumor material, and the tumor growth rate did not change during the subsequent mouse passages. Growth of PC-82 tumor tissue on female and castrated male mice did not occur. Castration of tumor-bearing mice resulted in a cessation of tumor growth, after which the tumor volume decreased 50 +/- 27 per cent within 6 weeks after castration. Hormone-independent regrowth of the tumor tissue was not observed after long-term withdrawal of androgens. After a period of 10 weeks following tumor growth arrest, administration of testosterone almost directly resulted in regrowth of the tumor. Hormones, testosterone and estradiol, were administered by silastic implants. Intact male nude mice were shown to have highly fluctuating levels of testosterone. Implantation with testosterone resulted in constant levels of circulating testosterone, which could be maintained for at least 10 weeks, while the mean concentration of plasma testosterone was not different from that in control male mice. The doubling time of tumors grown on testosterone-substituted intact female and intact and castrated male mice was significantly shorter than that of tumors grown on intact male mice. Histologically the tumors grown on androgen-substituted mice were similar to those grown on untreated mice; the mitotic index, however, was much higher in the testosterone treated animals. Implantation of intact male mice with estradiol suppressed plasma testosterone to a mean level of 1 ng. per ml. and prevented the growth of PC-82 tumor tissue almost completely. Treatment of tumor-bearing mice with an estradiol implant following androgen withdrawal did not result in a further decrease of the tumor volume compared to the mice without additional estradiol implantation.

Effect of Hormone Treatment on Prostatic Acid Phosphatase in a Serially Transplantable Human Prostatic Adenocarcinoma (PC-82)

The influence of endocrine manipulation on the tissue concentration of prostatic acid phosphatase (PAP) was studied in the hormone dependent transplantable human prostatic tumor line PC-82. Tumor bearing nude mice were left intact, castrated or treated for a 5-day period with a subcutaneous implant containing testosterone or estradiol. The concentration of PAP in castrated mice was not different from that in the controls. The DNA content of PC-82 tumor tissue obtained from 5-day castrated animals was significantly lower than that of tissue from intact animals. Therefore the concentration of PAP in tissue from castrated mice was significantly elevated when expressed per mg. of DNA (p less than 0.05). Treatment of the mice with testosterone or estradiol did not affect the PAP concentration in the tumor tissue. A significant correlation was observed between the concentration of PAP in the serum and the tumor burden of the mice. Long-term withdrawal of androgens resulted in a decrease of the concentration of PAP in the serum, as well as in a decrease of the tumor burden. The concentration of PAP in the tumor tissue remaining after castration of these animals was not significantly different from that in controls. The present data from the tumor line PC-82 do not support the hypothesis that the concentration of PAP in prostatic tumor tissue is controlled by androgens, but are in agreement with the concept that the level of PAP in plasma is related to the tumor mass.

The cytocidal effect of high energy shock waves on human prostatic tumour cell lines

This report describes the effect of high energy shock waves (HESW) generated by a Siemens Lithostar on four human prostatic carcinoma cell lines in vitro. The effects of temperature, shock wave energy, cell density and the number of HESW were investigated. Pressure measurements were carried out in the focus of the lithotriptor and inside test tubes that were placed in the focus. Direct cell kill was inversely related to temperature, whereas a linear relationship was found with shock wave energy. Cell kill appeared to be independent of cell density. All four cell lines were sensitive to the treatment with HESW, but displayed a different dose-response pattern. In vitro treatment of PC-3 cells retarded their growth upon injection into nude mice. It is concluded that human prostatic tumour cells are killed by HESW. Therefore, HESW could be of potential value in tumour treatment.

Glycosaminoglycans and other sulphated polysaccharides in calculogenesis of urinary stones

SummaryNaturally occurring glycosaminoglycans (GAGs) and other, semisynthetic, sulphated polysaccharides are thought to play an important role in urolithiasis. Processes involved in urinary stone formation are crystallization and crystal retention. Oxalate transport and renal tubular cell injury are determining factors in these processes. In this article experimental results concerning the possible mechanisms of action of GAGs and other sulphated polysaccharides are reviewed. GAGs are inhibitors of crystal growth and agglomeration and possibly also of nucleation. They can prevent crystal adherence, correct an abnormal oxalate flux and prevent renal tubular cell damage.

Quantitation of polyamines in cultured cells and tissue homogenates by reversed-phase high-performance liquid chromatography of their benzoyl derivatives

A rapid and simple method, originally described by Redmond and Tseng [J. Chromatogr., 170 (1979) 479] was applied to the analysis of di- and polyamines in cultured human tumour cells and human tumour xenografts. Optimization of the procedures and evaluation of the characteristic features of the assay are described. The (modified) procedure employs precolumn derivatization with benzoyl chloride, extraction of the derivatives by chloroform, separation by reversed-phase high-performance liquid chromatography under isocratic conditions and detection by ultraviolet absorbance measurement at 229 nm. The complete analysis was accomplished within 10 min per sample. The detection limit was ca. 1 pmol. The intra- and inter-assay coefficients of variation were 2.5-4.4% and 3.4-13.1%, respectively. The presence of well known inhibitors of polyamine biosynthesis, such as DL-alpha-difluoromethylornithine and methylglyoxal bis(guanylhydrazone), did not interfere with the assay, and disturbance by cyclohexylamine could be avoided by changing the polarity of the mobile phase. The method proved to be very suitable because it is rapid, simple, requires a minimum of sample pretreatment, and still provides sufficient sensitivity to quantitate polyamines in relatively small amounts of cells (10(5) cells) or tumour tissues (less than 1 mg), even after treatment with inhibitors of polyamine biosynthesis.