Human Testicular Cancer Research Articles

Modification of the Radiosensitivity of Human Testicular Cancer Cells by Simian Virus 40 Sequences

CRL7800 cells are of human testicular cancer origin and are sensitive to killing by ionizing radiation. After transfection with a plasmid expressing the T-antigen (pSC), cells show enhanced growth and an increased resistance to ionizing radiation. Cell cycle analysis reveals perturbation of a cell cycle checkpoint which, after irradiation, results in an increase in G2-phase arrest in CRL7800VA cells. These experiments demonstrate the modulation of radiation sensitivity and cell cycle arrest of human tumor cells by the introduction of viral genes.

The anti-tumour activity of ifosfamide on heterotransplanted testicular cancer cell lines remains unaltered by the uroprotector mesna.

Ifosfamide is clinically used in combination chemotherapy regimens for the treatment of patients with high-grade lymphomas, sarcomas and metastatic germ cell tumours. In order to reduce the oxazophosphorine-related urothelial toxicity, sodium mercaptoethane sulphonate (mesna) is used in different schedules following the administration of ifosfamide. The proposed mechanism of mesna activity is the binding of toxic oxazaphosphorine metabolites such as acrolein in the urine of the patients. Since an influence of mesna on ifosfamide anti-tumour activity is controversial, the current study has used xenografts from two human testicular cancer cell lines heterotransplanted into nude mice to study the anti-tumour activity of ifosfamide in combination with different dosages and schedules of mesna. In both human testicular cancer cell lines, H 12.1 and 2102 EP, ifosfamide demonstrated anti-tumour activity as a single agent. No reduction in ifosfamide activity was observed with the application of mesna at a dose range from 50% to 200% of the ifosfamide dose. Furthermore, the application of mesna before and 3 h after ifosfamide, a schedule used in many clinical protocols because of the short half life of mesna, not only maintained high ifosfamide anti-tumour activity but also seemed to be associated with the lower systemic and urothelial toxicity of ifosfamide therapy compared with ifosfamide given alone. In conclusion, the experimental in vivo system using human heterotransplanted testicular cancer cell lines confirms the significant anti-tumour activity of ifosfamide in malignant germ cell tumours and demonstrates that mesna does not impair ifosfamide anti-tumour activity in this model. These results are most likely transferable to the use of mesna in patients with metastatic testicular cancer.

Modulation of cytostatic drugs by nifedipine in two heterotransplanted human testicular-cancer cell lines differing in their sensitivity to standard agents.

Drug resistance is an important clinical problem in testicular cancer patients with relapsed or refractory disease after first-line chemotherapy. Here we report that the relative reduction in tumour volume in nude mice heterotransplanted with either H 12.1 or H 23.1 human testicular cancer cell lines was significantly increased by addition of the calcium antagonist nifedipine to the maximum tolerated dose (MTD) of cisplatin (DDP). The mean reduction in relative tumour volume at day 30 (rVR) reached statistical significance for both cell lines following combination therapy of DDP with nifedipine compared to DDP alone (55 +/- 7% versus 12 +/- 4% for H 23.1 and 60 +/- 9% vs. DDP with nifedipine has also been confirmed in H 12.1 cells in vitro. However, in vivo, this combination was associated with a concordant increase in therapeutic toxicity. In contrast, no improvement in in vivo anti-tumour activity was obtained by combining similar dose-schedules of nifedipine with the MTD of epirubicin, or with MTDs of vinblastine or etoposide. These results are in agreement with our immunohistochemical finding that H 12.1 and H 23.1 do not over-express the Pgp 170 glycoprotein which mediates the multiple drug resistance (MDR) phenotype and involves both anthracyclines and vinblastine, but not DDP. We conclude that another Pgp-MDR modulator, nifedipine, is able to increase the anti-tumour activity of DDP in vivo and in vitro via a specific but as yet unknown mechanism, which is most likely not MDR-related. However, the increased anti-tumour activity is, in vivo, associated with a considerable increase in overall toxicity. Further studies are necessary to decrease therapeutic toxicity, before clinically relevant models for modifiers of DDP-resistance could possibly be applied to patients.

NO GROWTH-STIMULATION OF HETEROTRANSPLANTED HUMAN TESTICULAR CANCER CELL-LINES BY RECOMBINANT HUMAN GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR (GM-CSF)

Hematopoietic growth factors such as granulocyte (G)- and granulocyte-macrophage (GM) colony-stimulating factor (CSF) are currently used as adjunct treatment for cancer chemotherapy in order to ameliorate neutropenia. The current investigation presented here focused on the in vivo effects of GM-CSF on two heterotransplanted human testicular cancer cell lines in a nude mouse model. GM-CSF was applied to tumor bearing mice in a dose range of 3 x 10(5) to 3 x 10(7) U/kg per day i.v. for 14 consecutive days. For all concentrations of GM-CSF a reduction of the tumor volume compared to untreated tumor bearing control mice was noted. The largest retardation of tumor growth was seen at the end of the experiments at day 30 with 3 x 10(7) U/kg of GM-CSF with a relative tumor volume of 72% +/- 8% for cell line H 12.1 and 73.5 +/- 9% for cell line H23.1 in comparison to untreated tumor bearing control mice. No side effects of GM-CSF application such as weight loss or fever were observed. Serum tumor necrosis factor (TNF) levels in mice treated with GM-CSF were considerably lower compared to untreated tumor carrying control animals. No growth stimulation of heterotransplanted human testicular germ cell tumors by GM-CSF has been observed in this nude mice tumor model. Further studies will have to demonstrate, whether GM-CSF is even possibly able to exhibit an antitumor effect. With respect to tumor cell kinetics the clinical use of GM-CSF in the treatment of patients with testicular cancer can be regarded as safe.

Establishment and characterization of a new human testicular germ cell tumor cell line (TCam-2)

A new human testicular cancer cell line (TCam-2) was established. The original material of TCam-2 was a primary lesion of a left testicular seminoma (typical pure type seminoma) from a 35 aged male patient. TCam-2 produced neither AFP nor beta-HCG, It showed strong immunoreactivities for 5G9 (anti testicular cancer MoAb), 4B3 (anti PTHrP MoAb) and PALP (placental alkaline phosphatase). The chromosomal analysis revealed 92 modal number and loss of Y chromosome. Histochemical, morphological and chromosomal analysis supported that TCam-2 is from classical seminoma. TCam-2 was transplanted subcutaneously to the back of 6 weeks old scid mice (CB-17 strain), and grew a classical seminomatous tissue.

Chromosome 12 in human testicular cancer: Dosage changes and their parental origin

Cytogenetically, a marker chromosome interpreted as i(12p) is present in most testicular tumors of germ cell origin. In this study, 22 patients with testicular germ-cell tumors were investigated by Southern blot hybridization to characterize changes in chromosome 12. In comparison with normal DNA, tumor DNA of 18 patients showed increased dosages of 12p accompanied by a comparable or smaller increase or no change in the dosage of centromeric sequences of chromosome 12. A likely interpretation was that most testicular tumors had one or several isochromosomes for 12p that were formed by somatic division of the centromere and that the points of breakage and reunion in the centromeric region were different in different tumors. Allelic 12p fragments showing increased intensity were paternal in four and maternal in three of seven informative cases. Thus, there was no evidence of sex-limited parental imprinting. Furthermore, the observed patterns of allelic fragments suggested that the marker was an i(12p) formed by sister chromatids of one homolog number 12 rather than the result of interchange of genetic material between different homologues.

Oncogenes in human testicular cancer: DNA and RNA studies

Oncogene dosage and expression were studied in 16 testicular neoplasms, 14 of germ cell and two of non-germ cell origin. In comparison with normal DNA, tumour DNA of a total of eight patients (seven with germ cell neoplasm and one with testicular lymphoma) showed increased dosages of KRAS2, PDGFA, EGFR, MET and PDGFB. The most frequent (occurring in six tumours) and prominent (up to 3-4-fold) increases were detected in the dosages of KRAS2 (on chromosome 12p) and PDGFA (chromosome 7p), relative to a reference locus from chromosome 2. Importantly, there was a similar increase in 12p dosage in general in these tumours, suggesting the presence of the characteristic isochromosome 12p marker. On the contrary, possible 7p polysomy (assessed by molecular methods) did not explain the PDGFA (or EGFR) changes in all cases. NRAS, MYCN, CSFIR, MYB, MYC, ABL, HRASI, TP53, and ERBB2 did not reveal any consistent alterations in tumour DNA. In RNA dot blot assays the expression of KRAS2, PDGFA, EGFR, or MYC was generally not increased in the tumour samples when compared to that in normal testicular tissue of the same patients although there was interindividual variation in mRNA levels. It thus appears that while oncogene dosage changes occur in a proportion of testis cancers, they are often part of changes in large chromosomal regions or whole arms and are seldom accompanied by altered expression.

P53 protein alterations in human testicular cancer including pre-invasive intratubular germ-cell neoplasia.

Expression of the p53 oncoprotein was examined in a wide range of primary human testicular germ-cell tumours using a new mouse monoclonal antibody (MAb) BP53-11 raised and characterized in this study, in parallel with a polyclonal rabbit antiserum CM-1. Immunohistochemistry on paraffin sections showed positive nuclear reaction in at least a fraction of malignant cells in 90 (84%) out of 107 cases studied. Aberrant accumulation of the p53 protein was found among testicular tumours of all major histological types, although generally a higher percentage of positive cases and a higher proportion of p53 over-expressing nuclei within individual lesions was observed in embryonal carcinomas when compared with seminomas. The typical heterogeneous staining pattern characteristic of histological specimens was also found in a cultured cell line derived from a human embryonal carcinoma. In contrast to immunohistochemically undetectable levels in normal testes and morphologically normal tissue areas in the tumour-bearing testes, the accumulation of the p53 protein was clearly identified in a high proportion (59% of cases) of the pre-invasive lesions with positive atypical intratubular germ cells often found in the tissue adjacent to invasive tumours. Altered expression of the p53 protein is therefore a unifying feature of the majority of invasive male germ-cell tumours and the change resulting in high levels of p53 appears to be a relatively early step in the human testicular cancer pathogenesis.

DNA methylation changes in human testicular cancer

We previously reported and X/Y imbalance with a relative excess of X- and a relative deficiency of Y-chromosomal DNA in three out of nine testicular tumors of germ cell origin. To study the implications of those changes the methylation status of DNA from seven of the tumors was explored by HpaII/ MspI analysis. The 5′ regions of the hypoxanthine phosphoribosyltransferase (HPRT) and the phosphoglycerate kinase (PGK) gene loci inhibited main patterns suggestive of active X chromosomes in the tumors. However, a minority of the HPRT loci of one teratocarcinoma with an increased dosage of the X chromosome, as well as one additional teratocarcinoma, revealed patterns analogous to inactive X chromosomes in females. Using probes from several chromosomes it was subsequently found that the teratocarcinoma tumors ( 3 3 ) were characterized by generalized hypermethylation. On the contrary, the seminomas showed variable hypomethylation ( 4 5 ) or virtually complete demethylation ( 1 5 ). The seminoma with the most extensive hypomethylation was disseminated (stage III), whereas the other seminomas were local (stage I). These findings suggest that DNA methylation may play a role in the developmental pathways leading to different histologic types of testicular tumors of germ cell origin. The HPRT results imply that the consequences of extra X chromosomes - a frequent finding in testicular tumors - may be modulated by mechanisms, such as DNA methylation, that control gene activity.

Altered dosage of the sex chromosomes in human testicular cancer: A molecular genetic study

Thirty-one males with testicular germ-cell tumors were studied by Southern hybridization using X- and Y-chromosome-specific probes as well as a pseudoautosomal probe. Densitometric analysis showed changes in the relative dosage of Y-chromosomal fragments in tumor DNA from 12 out of 31 patients (39%) as compared to normal DNA from the same patients. In 11 tumors the relative intensity ratios of Y-chromosome-specific fragments had decreased from the normal value of 1 to values between 0 and 0.77. An increase in the Y-chromosomal dosage was observed in 1 case. The entire Y chromosome was apparently involved in most patients but 2 tumors revealed regional variation. Tumor DNA of 2 patients with Y-chromosomal deficiency showed a concomitant increase in the X chromosomal dosage. The pseudoautosomal region that is shared by both sex chromosomes was involved in a total of 8 tumors (26%), 2 of which did not show any obvious dosage changes with probes detecting strictly X- or Y-chromosome-specific fragments. Autosomal alterations in the present tumor series have been described. A dosage change involving the sex chromosomes accompanied loss of heterozygosity at loci in 3p or 11p in 10 tumors out of 15 (67%). Seminomas tended to be affected more often than non-seminomas by either type of alteration. Our results indicate that changes in the sex chromosomes occur in a substantial proportion of male germ-cell tumors and, together with other defects, may constitute an important step in tumor development and/or progression.

Dipyridamole-Cisplatin Potentiation: Enhanced in Vivo Cytotoxicity in Xenograft Models of Human Testicular and Bladder Cancers

The antitumor efficacy and host toxicity of dipyridamole (DP), methotrexate (MTX) and cisplatin (CDDP) alone and combined were evaluated in a nude mouse supported human bladder cancer model. Single agent post treatment tumor volume growth ratio [TGR] values of DP, MTX and CDDP were 97%, 65% and 49% of control. While the MTX/DP combination produced only mild cytotoxic enhancement, CDDP/DP and CDDP/MTX/DP reduced TGR to 20% and 17%, respectively. A second multi-dose evaluation of CDDP/DP using human testicular carcinoma in this model also showed a CDDP dose-dependent response with achievable complete tumor regression. Host toxicity was not substantially increased by DP. DP would appear to be effective in vivo as a chemosensitizer of CDDP; it may enhance the therapeutic efficacy of CDDP in a variety of tumors. (J. Urol., 144: 1004–1009, 1990)

Comparative activity of four anthracyclines against heterotransplanted germ cell tumor lines

Though the majority of patients with metastatic nonseminomatous germ cell tumors can be cured by modern combination chemotherapy, for those patients who do not respond to standard therapy additional drugs are needed. The activity of three new anthracycline derivatives, 4-epidoxorubicin, THP-doxorubicin and mitoxantrone against two established human testicular cancer cell lines in comparison to doxorubicin and to cisplatin, vinblastine, bleomycin and ifosfamide was studied in a xenograft model. All drugs were given at equitoxic doses. There were no differences in antitumor activity between the four anthracycline derivatives. In line H 12.1, which is very sensitive to the standard drugs cisplatin, vinblastine, bleomycin and ifosfamide, all four anthracycline derivatives were inferior to these agents. In contrast, in line H 23.1, where all four standard agents showed a significant lower antitumor activity when compared to line H 12.1, the anthracyclines preserved their activity, indicating a lack of cross resistance. Thus the anthracycline derivatives seem to be inferior to the standard drugs as first line treatment but because of apparent lack of cross resistance they deserve further evaluation in refractory germ cell tumors.

Activity of cytostatic drugs in two heterotransplanted human testicular cancer cell lines with different sensitivity to standard agents

Two established human testicular cancer cell lines were used in a mouse xenograft model to assess the antitumour activity of 15 anticancer agents. Line H 12.1 was highly sensitive to cisplatin, bleomycin and vinblastine, resembling non-pretreated testicular tumours, whereas line H 23.1 showed resistance to cisplatin and vinblastine, comparable to tumours with acquired or intrinsic drug resistance. In line H 12.1 several drugs were highly active, including cyclophosphamide, ifosfamide, nimustine and vincristine; carmustine, vindesine, doxorubicin, epidoxorubicin, pirarubicin, mitoxantrone, carboplatin and iproplatin had only moderate activity. In line H 23.1 only cyclophosphamide, ifosfamide, nimustine, vincristine and bleomycin had antitumour activity. These two cell lines represent a useful model for preclinical evaluation of new agents with presumed activity in testis cancer.

Molecular studies of the sex chromosomes in human testicular cancer: Pronounced changes in X and Y chromosome dosage in some tumors

Nine males with testicular germ cell tumors were studied by Southern blotting using probes recognizing different regions of the X and Y chromosomes. In the tumors of three patients, an imbalance was noted with a relative deficiency of DNA of Y-chromosomal and a concurrent excess of that of X-chromosomal origin. The X:Y signal ratios were 4, 4, and 2, respectively, in tumor DNA relative to normal DNA, and the ratios of Y to an autosomal locus were 0.4, 0.6, and 0.7, respectively, in the same tumors. Several loci on both arms of the Y chromosome were similarly involved. No structural abnormalities of Y chromosome DNA could be demonstrated. The X/Y change occurred in two of the three patients with nonseminomatous tumors and in two of the three patients with metastatic disease. To account for the results, two alternative models are discussed: first, loss of the Y chromosome and increase in X chromosome number in some but not all tumor cells; second, polyploidization with one Y chromosome and several X chromosomes.

Expression of myc family oncogenes in primary human testicular cancer

In order to understand the role of myc family genes (c-myc, N-myc and L-myc) in the development and progression of human testicular cancer, we have analyzed the expression of myc family genes in three different types of primary human testicular cancer (seminoma, embryonal carcinoma and teratocarcinoma) and normal testis using Northern blot analysis. Expression of N-myc gene, which is usually limited in the neoplasms derived from neuroectoderm, was detected in seven out of ten cases of seminomas and two out of two cases of embryonal carcinomas. Gene amplification was not observed in these cases. Expression of N-myc gene was not detected in teratocarcinomas and normal testes. Expression of c-myc gene was observed in seminomas, embryonal carcinomas, teratocarcinomas and normal testes, but specific expression of c-myc gene was not seen in these cancers and normal testes. Expression of L-myc gene was not detected in all cases examined in our studies. Since N-myc gene expression was observed only in undifferentiated testicular neoplasm, such as seminoma and embryonal carcinoma, its expression may be positively related to the development and progression of special types of human testicular cancer.

Isolation and purification of placental type alkaline phosphatase from a seminoma.

An alkaline phosphatase (EC 3.1.3.1) of the placental type was isolated from a seminoma type of human testicular cancer tissue and was purified to homogeneity by sulfate-mediated chromatography on a column of Cibacron Blue Sepharose 4B. The purified enzyme had a specific activity of 40.6 kU per gram of protein and was obtained in a yield of 37%. The purification procedure used was simple and economical, and may be used to purify alkaline phosphatase isoenzymes from other cancer tissues. This is the first report of the purification of the enzyme in seminoma. Inhibition studies suggest that this enzyme is a Nagao variant rather than the Regan type reported in several cancer tissues.

The influence of glucocorticoids on the proliferation of glucocorticoid receptor positive human testicular cancer cell lines

The influence of glucocorticoids on the proliferation of glucocorticoid receptor positive human testicular cancer cell lines

Pregnancy-Associated Plasma Protein-a Concentrations in men with Testicular and Prostatic Tumors

Pregnancy-associated plasma protein-A (PAPP-A) is a macromolecular glycoprotein produced during pregnancy by the trophoblast and the decidua. It is not specific to pregnancy, since immunoreactive PAPP-A is in the circulation of normal women and men and in seminal fluid. Immunohistochemically, PAPP-A is localized in some Leydig cells, in the epididymis, in the rete testis, and in seminal vesicles. This study was conducted to learn whether circulating levels of PAPP-A can be used as a marker for the monitoring of human testicular cancer. PAPP-A was increased only in certain nonseminomatous germ cell tumor [malignant teratoma undifferentiated (MTU) + seminoma, malignant teratoma intermediate (MTI)], but the overlap with the normal values was too big for PAPP-A to have any clinical significance. After orchidectomy or prostatectomy, PAPP-A decreased but the levels were were similar to those of normal men. In men, PAPP-A can be produced by certain nonseminomatous germ cell tumors, but its origin in disease-free males is probably not the testis or the prostate.

DNA flow cytometry in human testicular cancer

DNA flow cytometry revealed aneuploid tumour stemlines in 19 of 20 primary testicular cancers without significant difference of the ploidy values between seminomas and non-seminomas. In 7 of 8 analyzable histograms the S-phase activity was 22–51%. A metastatic mature teratoma had 6% cells in S-phase. These results support the clinical observation that testicular cancer is usually a rapidly growing human tumour. The high percentage of aneuploidy in testicular cancer may be of clinical value in the diagnosis of this malignancy.

Characteristics and chemotherapeutic sensitivity of a human testicular cancer grown in artificially immunosuppressed mice.

Seven human testicular tumors were transplanted into artificially immunosuppressed mice. Two of them grew progressively (TT2 and TT6) and a serially transplantable line was developed from TT2. The xenografts maintained only the embryonal carcinoma components of originally mixed (embryonal cell carcinoma and choriocarcinoma) donor tumor. Although the histology did not change remarkably with passages, the xenografts lost their capacity to express human choriogonadotropin and alpha-fetoprotein. The latency period shortened, the growth rate remained similar with subsequent transplantations. The tumor cells of the TT2 line presented the human character according to chromosome analysis and were built up of two subsets of cells with a different DNA index estimated by flow cytometry. The embryonal cell carcinoma line was highly sensitive to CY and cisDDP. PVB combination was also effective, although the tumor growth inhibition proved to be only temporary.