Biomedical subjects

J C Romijn

Publications and source records attributed to J C Romijn.

At least 73 records · Page 4Linked to original sources

PubMed1989

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.

Amino Acid Sequence↗

PubMed1989

Cytogenetic characterization of several androgen responsive and unresponsive sublines of the human prostatic carcinoma cell line LNCaP.

The cytogenetic evolution of the prostatic adenocarcinoma cell line LNCaP was investigated during long term in vitro culture. Study of five different sublines demonstrated that the original karyotype was well preserved in all sublines, with respect to the chromosome number as well as to the primary markers. All sublines showed additional, subline specific secondary marker chromosomes. Comparison of these markers in androgen responsive and nonresponsive sublines showed rearrangement of the short arm of chromosome 8 in both unresponsive sublines. The breakpoints were in 8p21 and 8p23, respectively, resulting in deletion of the 8p23----pter region in both sublines. In contrast, the hormone responsive sublines did not show any aberrations in chromosome 8. Review of published karyotypes of patients and cell lines seems to support our finding of partial deletion of 8p in androgen unresponsive prostate tumor cells.

Androgens↗

PubMed1989

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.

Cell Line↗

PubMed1989

Human renal cell carcinoma in the six day subrenal capsule assay.

The applicability of the subrenal capsule assay (SRCA) as a method for chemosensitivity testing of human renal cell carcinoma (RCC) was assessed. Tissue fragments of about one mm3. in size, obtained from patients who had undergone nephrectomy because of renal cell carcinoma (RCC), were implanted in preirradiated mice. The change of the size of the implanted tissue as well as the histology of the tissue were determined six days later. Untreated control tumors were used to determine the evaluability of the individual assays using criteria based on 1) tumor growth (an increase in tumor size or a decrease not exceeding 0.05 mm.) and 2) tumor histology (presence of carcinoma cells in at least 70% of the tissues). The first criterion was fulfilled in 20/24 (83%) of the renal tumors; tumor histology was satisfactory in 9/24 (38%) of the tumors. Taken together, only 9/24 assays were considered to be evaluable. Discrepancies between measurement and histologic appearance were also found for individual tumors and for treated tumors. Due to the lack of correlation between measurement and histology change in tumor size therefore cannot be considered a reliable parameter for assessing viability or drug activity.

Animals↗

PubMed1988

Molecular cloning and characterization of novel prostate antigen cDNA's.

Three different prostate antigen cDNA's were isolated from a PC 82 prostate tumor cDNA library. PA 75 has a size of 1.4 kb and contains the almost complete information for the 35 kD prostate antigen preproprotein. The 1.6 kb PA 525 cDNA lacks about 0.2 kb of the 3'-non coding region and contains an additional internal 0.4 kb fragment as a result of alternative splicing. PA 424 represents a 0.6 kb variant of PA 75. It contains a 0.15 kb internal fragment and a poly(A) tail preceded by an AAGAAA motive at the 3'-end. The predicted protein products of PA 525 and PA 424 will be different from PA 75 at the C-terminal end. In RNA preparations of two human prostate tumors (PC 82 and PC EW) seven different prostate antigen transcripts can be detected ranging in size from 0.5 kb to 5.6 kb. PA 75 cDNA represents the major 1.5 kb mRNA. PA 424 correlates with a 0.9 kb transcript and PA 525 with a 1.9 kb mRNA species.

Amino Acid Sequence↗

PubMed1988

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.

Animals↗

PubMed1988

Application of the MTT assay to human prostate cancer cell lines in vitro: establishment of test conditions and assessment of hormone-stimulated growth and drug-induced cytostatic and cytotoxic effects.

In this study the usefulness of the MTT assay for the quantitation of growth modulating effects on cultured prostate cancer lines (PC-3, PC-93, and LNCaP) was investigated. The MTT test is based on the enzymatic reduction of the tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide++ +] in living, metabolically active cells but not in dead cells. The reaction is carried out in situ in multiwell plates, and the reaction product, a purple-colored formazan soluble in dimethylsulfoxide, is measured colorimetrically, using a multiwell plate reader. Optimal test conditions were established for each of the cell lines used. With the hormone-sensitive cell line LNCaP, and stimulatory effect of the synthetic androgen R1881 was demonstrable by the MTT test. A sharp optimum occurred at a concentration of 10(-10) M R1881. Treatment of cells of either cell line with antineoplastic agents resulted in a dose-dependent reduction of MTT converting activity, reflecting the impaired survival of the drug-treated cells. Good correlations of the results obtained with the MTT-test, as compared with a thymidine incorporation assay or with direct DNA measurements, were observed. As the MTT test offers a high degree of precision and is easy to do, it is suitable for the purpose of (large-scale) chemosensitivity testing. Moreover, serial measurements might easily be performed in order to provide additional information on the mode of action of the drugs tested, i.e., to discriminate between cytostatic and cytotoxic drug effects.

Androgens↗

PubMed1988

In vitro antiproliferative efficacy of interferon-alpha, -gamma and tumor necrosis factor on two human renal tumor xenografts.

The in vitro antitumor activity of recombinant alpha- and gamma-Interferon as well as recombinant Tumor Necrosis Factor alpha was tested on renal cell carcinoma xenografts using the double layer soft agar method. Using this assay, the effect of the drugs on the clonogenic potential of tumor cells in soft agar was determined and used as an indication for the antiproliferative capacity of these drugs. There appeared to be a differential response of the tested xenografts towards these drugs in a dose dependent way. However, when used in combination in most cases an additive or synergistic effect was observed. Even though again the response was differential, the combination of alpha-Interferon (10 ng/dish) and Tumor Necrosis Factor (100 ng/dish) resulted in a complete inhibition of colony formation in soft agar for both tumor lines. We conclude that there is a direct effect of alpha-Interferon, gamma-Interferon and Tumor Necrosis Factor on renal cell carcinoma xenografts. In combination drug tests the effect of alpha-Interferon and Tumor Necrosis Factor was strongly synergistic. The implication of these studies for in vivo use of these drugs remain to be established.

Animals↗

PubMed1988

Cytogenetic characterization of an established xenografted prostatic adenocarcinoma cell line (PC-82).

Detailed cytogenetic analysis was performed of a xenografted human prostatic adenocarcinoma cell line PC-82. A direct preparation method was developed that yielded metaphases of good quality. Flow cytometric data and banding analysis of metaphases showed a near-tetraploid karyotype with 18 consistent marker chromosomes. As a result of the rearrangements involved, parts of chromosomes 2, 3, 4, 7, 9, 10, 15, 18, and 21 were homozygous, while regions on 2p, 13q, and 17q were apparently completely lost. In contrast to this, some regions on #2, #5, and, especially, on #1 were present in three or even four times the normal copy number. Comparison of affected chromosomes in PC-82 with all data available on prostatic carcinoma showed chromosomes 1, 2, 3, 6, 7, 10, and 15 to be involved in rearrangements in over 50% of all prostatic carcinomas. When only data from primary prostatic adenocarcinomas (including PC-82) were taken into account it appeared that chromosomes 1, 7, and 10 were involved in all five primary tumors studied.

Adenocarcinoma↗

PubMed1988

In vivo and in vitro chemotherapy sensitivity testing for human kidney tumor lines: a comparative study.

In vivo and in vitro chemosensitivity testing has been applied to kidney carcinoma tumor lines. Serially transplantable tumors were implanted subcutaneously in nude mice and the animals were treated with cytostatic drugs. The results of this in vivo assay were compared with the results obtained with the subrenal capsule assay, the DNA precursor assay (3H-thymidine), and the colony-formation assay, utilizing the same tumor line in each case. Higher rates of resistant tumors were found in the in vivo assays than in the in vitro assays. The SRC assay and the DNA assay had the highest predictive value, as judged from comparison with the results obtained with the source tumor (human kidney carcinoma tumor line).

Animals↗

PubMed1987

Decarboxylated-S-adenosylmethionine excretion: a biochemical marker of ornithine decarboxylase inhibition by alpha-difluoromethylornithine.

In an attempt to define a biochemical marker of ornithine decarboxylase inhibition in humans, alpha-difluoromethylornithine hydrochloride (DFMO), an irreversible ornithine decarboxylase inhibitor, was infused i.v. in seven cancer patients over 10-day courses at doses of 10-90 g/day and 24-h urinary excretion of polyamines and decarboxylated-S-adenosylmethionine was determined before, during, and after treatment. DFMO produces marked increases in urinary decarboxylated-S-adenosylmethionine excretion, up to 84 times pretreatment values. This response appears to be time dependent, requiring several days to reach a maximum and lasting at least 4-5 days after stopping DFMO. In contrast, urinary excretion of the polyamines putrescine, cadaverine, spermidine, N1-monoacetylspermidine, N8-monoacetylspermidine, and spermine, were not consistently altered by DFMO. We conclude that urinary excretion of decarboxylated-S-adenosylmethionine represents a valid biochemical indicator of ornithine decarboxylase inhibition in humans, whereas urinary polyamines are of no value.

Aged↗

PubMed1987

Prostatic epithelium inhibiting factor (PEIF): organ specificity and production by prostatic fibroblasts.

The effects of prostate fibroblast conditioned medium on two prostate epithelial cell lines (PC-3, LNCaP) and on two non-prostatic cell lines (MCF-7, K562) was investigated. As prostate fibroblast conditioned medium exerts its main effect on DNA synthesis, 3H thymidine incorporation was monitored to measure factor activity. Conditioned media of all prostatic fibroblast lines investigated were inhibitory for PC-3, LNCaP and MCF-7. Conditioned medium of prostatic fibroblasts was clearly stimulatory for K562. Prostate specificity of production of PEIF was demonstrated by the fact that conditioned medium from skin fibroblasts proved to be stimulatory for PC-3. Inhibitory activity from conditioned medium as well as from a BPH homogenate was precipitated by 33-67% ammonium sulfate. These partly purified fractions were respectively five and ten times as active as "crude" conditioned medium. The physical nature of PEIF (protein or macroglycolipid) as well as the possible function (as a signal messenger between stroma and epithelium) is discussed.

Ammonium Sulfate↗

PubMed1987

Problems of pharmacokinetic studies on alpha-difluoromethylornithine in mice.

The pharmacokinetics of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of polyamine biosynthesis, were investigated in BALB/c (nude) mice after i.p. injection and after oral administration of radiolabeled drug. After i.p. injection the compound was rapidly cleared from the serum (t1/2 alpha = 14 min; t1/2 beta = 2.1 h) and from tissues such as muscle, liver and kidney (t1/2 alpha = 30-60 min; t1/2 beta = 2.1 h). DFMO concentrations were proportional to the administered dose (10-2000 mg/kg) in both serum and tissues. Oral administration of DFMO was carried out by dissolving the compound in drinking water at a concentration of 20 g/l. Studies on the distribution showed that DFMO did not accumulate preferentially in any particular tissue. An extremely wide variation in the dose actually achieved in different animals was observed; this ranged from 350 to 2800 mg/kg for a 14-h treatment period. A significant correlation (r = 0.83-0.92) between the dose of DFMO, calculated from the consumption of drinking water for each individual animal, and the DFMO concentrations in serum, muscle, spleen, liver and kidney was found. Similarly, it was shown that oral administration of DFMO during the daytime resulted in 10- to 15-fold lower levels than administration during the night. After discontinuation of treatment DFMO levels in serum and tissues decreased by 50% in approximately 6 h. From these results it is concluded that the optimal treatment schedule of mice with DFMO (or other drugs with similar pharmacodynamic properties) consists in a combination of oral administration via the drinking water and additional i.p. injection (during the daytime). Furthermore, the drug intake of the individual animals should be monitored to check whether the experimental requirements are actually fulfilled.

Administration, Oral↗

PubMed1987

Isolation and individual electrical stimulation of single smooth-muscle cells from the urinary bladder of the pig.

In contrast to striated muscle, measurements on strips of smooth muscle cannot be uniquely interpreted in terms of an array of contractile units. Therefore scaling down to the single-cell level is necessary to gain detailed understanding of the contractile process in this type of muscle. The present study describes the development of a method for isolating contractile single smooth muscle cells from pig urinary bladders. Contractile responses evoked by individual electrical stimulation were used as a measure of cell quality during development of the method. Responses were evaluated by measuring latency, contraction and relaxation times, as indicated by visible length changes, and stored on-line in a computer. Initial length, relative shortening and shortening speed were determined by measuring cell lengths in previously timed still video frames using a computer-controlled crosshair device. Increase of stimulus pulse duration resulted in improved responses, indicating that the observed shortening represented a physiological contractile response. Ultimately this method of evaluation was applied to two sets of cell preparations obtained by two different methods, one using only collagenase digestion, the other using mechanical manipulation as well. Both sets showed two main patterns of response to electrical stimulation: a pattern of contraction upon stimulation followed by enhanced contraction when stimulation was switched off (CK), and a pattern of contraction upon stimulation followed by relaxation when the stimulus was switched off (CR). The set of preparations containing the highest percentage of CR cells was found to be superior (i.e. greater initial length, shorter latency and contraction times, increased shortening and higher shortening speed). The method of isolation used for this set gives a high yield of contractile cells available for experimental use over a long span of time.

Animals↗

PubMed1986

Measurement of the survival of human tumor cells after implantation in athymic nude mice.

A method has been developed to determine the survival of human tumor cells after implantation in athymic nude mice. The method involves the labelling of cells of the prostatic cancer cell line PC-3 with tritiated thymidine after attachment of the cells to Millipore filter disks. The amount of radioactive label incorporated in the cells remained constant for at least 48 hr during in vitro incubation. After s.c. implantation of the filter disks in nude mice, however, the fraction of surviving cells, reflected by the amount of radioactivity persisting on the filters, rapidly decreased with time. Only a relatively small fraction (approx. 10%) of the cells survived for more than 24 hrs under these conditions. Pretreatment of the mice with cyclophosphamide (100 mg/kg, 24 hr prior to implantation) resulted in a 2-fold increase of tumor-cell survival. Likewise, the frequency of tumor takes after injection of PC-3 cells was enhanced following cyclophosphamide pretreatment. These results indicate that immune defense mechanisms might be partly (but not completely) responsible for tumor-cell rejection in nude mice. The technique described here might be a valuable tool for the rapid evaluation of possible effects of various types of pretreatment of the host animal on tumor cell survival and, thereby, on tumor growth.

Animals↗

PubMed1986

Characterization of monoclonal antibodies raised against the prostatic cancer cell line PC-82.

For production of monoclonal antibodies (McAbs), hybrid cells were prepared by fusion of spleen cells of BALB/c mice immunized with the human prostatic cancer cell line PC-82 and the P3-X6(3)Ag8.653 murine myeloma cell line. Supernatants of approximately 500 hybrid clones were screened for prostate specific antibodies using an ELISA on PC-82 cells. A selection of antibodies was further tested for their specificity on a large series of different tissues. A broad cross reactivity pattern was obtained. Most cross reactivity was with pancreatic tissue, kidney, and bowel. One antibody turned out to react with prostate stromal cells. Two McAbs (ER-Pr 1 and ER-Pr 2) reacted solely with prostatic epithelium. Monoclonal antibody affinity chromatography combined with SDS-PAGE showed that both antibodies were directed against a 35-kD protein. Immunoblotting revealed that this protein is identical to prostatic antigen (PA). The epitope detected by ER-Pr 1 and ER-Pr 2 was largely preserved after formalin-fixation of prostatic tissues which renders these antibodies very suitable for routine examination of tissue sections.

Animals↗

PubMed1986

Species-dependent differences of the biochemical properties of diamine oxidase.

Diamine oxidase (DAO) from tissues of mice, rats and humans showed different properties with respect to stability and kinetic parameters. DAO-activities in homogenates of rat or human tissues, but not of mouse tissues, rapidly decreased upon storage at -20 degrees C. The Km-value for putrescine was 90 microM in mouse kidney or intestine. In rats different Km-values were observed before (272 microM) and after freezing (102 microM). A similar effect was observed with DAO in human kidney (321 and 39 microM, respectively). Treatment of rats with heparin resulted in a depletion of intestinal DAO and the concomitant appearance of DAO in blood. The enzyme remaining in the intestine showed the lower Km-value.

Amine Oxidase (Copper-Containing)↗

PubMed1986

Phase I study of alpha-difluoromethylornithine and methyl-GAG.

alpha-Difluoromethylornithine (DFMO) is a potent inhibitor of the synthesis of putrescine (pu) and spermidine (sd) in some benign and malignant tissues. Intracellular deprivation of pu and sd has been shown to induce an enhanced uptake of polyamine-analogs such as methyl-GAG (MGBG). The purpose of this study was to investigate the tolerance and the toxicity of the combination of DFMO and MGBG. Thirty-six patients received 4 X 2 g of DFMO/day orally and every 2 weeks 250-500 mg/m2 of MGBG as a 2-hr infusion, starting on day 14. Besides the well known acute and late side-effects of methyl-GAG, dose-limiting toxicity consisted also of thrombocytopenia, leucopenia, dyspnea, hemolysis and jaundice. The maximal tolerated dose of MGBG for one course was 350 mg/m2 and for repeated courses 250 mg/m2, due to cumulative toxicity. Furthermore, after 8 weeks of continuous administration of DFMO 70% of the patients had a severe hearing loss, which was reversible after a treatment delay of 4-6 weeks. Since the hearing loss prohibited the continuous use of DFMO, two different schedules of intermittent DFMO-administration together with two different infusion periods of MGBG have been investigated in 15 patients. In none of these patients did hearing loss occur. The schedule of continuous administration of 4 X 2 g of DFMO/day orally for 21 days and 250 mg/m2 of MGBG as a 24-hr infusion on days 7, 14 and 21, repeated on day 42, was tolerated best. In 28 evaluable patients two partial remissions were seen. Pretreatment with DFMO significantly enhanced the toxicity of MGBG and the combination of both drugs produced side-effects not seen with either drug alone.

Adolescent↗