Nonrandom chromosome abnormalities in testicular and ovarian germ cell tumor cell lines

We report the karyotypic analysis of seven testicular and one ovarian germ cell tumor (GCT) cell lines, a number of which have previously been partially investigated. An i(12p) was found in each of the testicular GCT cell lines, while it was absent in the ovarian GCT cell line. Thus, our study extends to cell lines the observation from fresh tumor tissues that i(12p) is a highly nonrandom chromosomal abnormality of testicular GCT. Additional consistent nonrandom abnormalities in the testicular GCT cell lines included the following: del(1)(p22), del(1)(q21), i(1q), del(7)(q11.2), and del(12)(q14). The del(12)(q14) abnormality was identified in five of the cell lines investigated. This observation, together with previous detection of this marker chromosome in fresh tumor specimens by us and others, suggests that loss of genetic material on 12q may represent a primary change associated with malignant transformation of testicular germ cells. As reported in a previous study, a t(15;20)(p11;q11) translocation was identified in the ovarian GCT cell line. Interestingly, it also was seen in one testicular GCT cell line. In addition, a der(15)t(15;20)(p11;q11) marker chromosome was identified in two other testicular GCT cell lines. Thus, this reinvestigation of GCT cell lines has resolved the discrepancy regarding the occurrence of i(12p) in fresh tumors versus established cell lines and identified additional nonrandom abnormalities of potential importance to the development of GCTs.     

Identification of genomic changes associated with cisplatin resistance in testicular germ cell tumor cell lines

Since the introduction of cisplatin into the clinic, the treatment of patients with a variety of solid tumors including testicular germ cell tumors, ovarian and lung cancers, has dramatically improved. One of the main causes for therapeutic failure in these malignancies is the development of drug resistance. Testicular germ cell tumors (TGCTs), the most common malignancy in young men, exhibit extreme sensitivity to cisplatin-based chemotherapy, making them an ideal model for investigating the mechanisms of cisplatin chemo-sensitivity and resistance. TGCT development and pathogenesis have been well studied but little is known about the genetic background in chemo-resistant cases. We investigated genomic differences between three TGCT parental cell lines and their cisplatin resistant derivatives. Using 10K single nucleotide polymorphism (SNP) microarray analysis, we identified two small chromosomal regions with consistent copy number changes across all three pairs of resistant cell lines. These were an 8.7 Mb region at 6q26-27, which displayed consistent copy number gain and a 0.3 Mb deletion involving 4 SNPs at 10p14. Both the chromosomal gain and loss were confirmed by fluorescence in situ hybridization. The significance of these regions should be further investigated as they may contain key genes involved in the development of chemo- resistance to cisplatin-based treatment in TGCTs and other cancers.     

Production of interferon by human tumor cell lines

Summary Fourteen continuous human cell lines, including nine derived from tumors and five from non-neoplastic tissues, produced interferon in response to induction with bluetongue virus (BTV), Newcastle disease virus (NDV), and poly(I) · poly(C) complexed with DEAE-dextran. The seven best interferon-producing cell lines (one from a melanoma, five derived from carcinomas, and one SV40-virus-transformed kidney cell line) responded to at least one of the viral inducers with yields of interferon over 1000 units/ml. Because the HT-1376 bladder carcinoma cell line produced high yields of interferon in this survey, and is easily propagated, the optimal conditions for interferon production were investigated, using BTV as the inducer. Interferon yields in 59 inductions over a period of about two years consistently fell within a 6-fold range, and had a geometric mean titer of about 2700 reference units (RU)/ml, representing the production of about 3 RU/10³ cells. This yield is comparable to mean titers of 1 to 10 RU/10³ cells obtained by others with human leukocytes, foreskin cell strains, or the Namalva lymphoblastoid cell line. UV-inactivated BTV at a multiplicity corresponding to 10 PFU/cell was as effective an inducer in the HT-1376 cell line as the fully infectious virus at a multiplicity of 1 PFU/cell. The interferon produced by the HT-1376 epithelial cell line has characteristics similar to the interferon induced by poly(I) · poly(C) in human diploid fibroblasts.These studies clearly demonstrate that many different types of tumor-derived cells have the capacity to produce interferon, and that some equal or surpass the efficiency of diploid cells.With 1 Figure     

Synthesis and antiproliferative activity of new coumarin-based benzopyranone derivatives against human tumor cell lines

The synthesis and antiproliferative activity of new coumarin-based benzopyranone derivatives containing basic amino side chain are described. The cytotoxicities against A549 and MCF-7 human cancerous cell lines were determined after 24, 48, 72 h drug exposure employing MTT assay at concentrations ranging from 0-100 μM. The antiproliferative activities of these compounds were compared to tamoxifen (TAM), 4-hydroxytamoxifen (4-OHT), raloxifene (RAL), 17β-estradiol (E2) and Diethylstilbestrol (DES). In vitro results indicated that compounds 10 and 12 were the most potent showing significant inhibitory activities against these cell lines. Furthermore, their antiproliferative activity against MCF-7 human breast cancer cell line is comparable to that of TAM, RAL and 4-OHT.     

Overexpression of genes on 16q associated with cisplatin resistance of testicular germ cell tumor cell lines

Testicular germ-cell tumors (TGCTs) show exquisite sensitivity to cisplatin-based chemotherapy, and therefore this is considered a good model system for studying the mechanism of chemotherapy resistance. Although the genetic alterations related to TGCT have been well studied, little is known about the genetic basis of chemotherapy resistance, which occurs in a small proportion of TGCTs. In this study, we investigated genomic and expression differences between three cisplatin-sensitive and their paired cisplatin-resistant lines using combined whole-genome screen approaches. Comparative genomic hybridization (CGH) analysis on chromosomes revealed genetic differences between the resistant and parent cell lines in each pair, but did not show any consistent chromosome changes in all three lines. Microarray CGH analysis generated some additional information of DNA copy number gains and losses including some important oncogenes, tumor-suppressor genes, and drug-resistance-related genes. However, no consistent genomic region changes were found in the three cell lines. Interestingly, when comparative expressed sequence hybridization, a technique for gene expression profiling along chromosomes, was applied, we discovered a consistently overexpressed chromosomal region in all three resistant lines compared with their parent lines. The minimum overlapping chromosomal region is at 16q22-23. Further definition of genes in this chromosomal region will aid our understanding of the mechanism of cisplatin resistance and may offer novel therapeutic targets.     

Ovarian germ cell tumor evolving to myelodysplasia

We present the case of a 14-year-old girl in whom a myelodysplastic syndrome was diagnosed 9 months after surgical resection and chemotherapy for an ovarian germ cell tumor. Cells from her marrow were characterized by trisomy 8 and an isochromosome 12p, a marker that appears to be unique to germ cell tumors. The presence of the same two anomalies in her original tumor was demonstrated by fluorescence in situ hybridization study of interphase cells in paraffin-embedded tissues and thus provided strong evidence that the two neoplasms had a common clonal origin. © 1993 Wiley-Liss, Inc.     

Microinvasive germ cell tumor of the testis

Microinvasive germ cell tumor (MGCT) consists of a limited number of malignant germ cells in the intertubular tissue of the testis. The cells have large nuclei, prominent nucleoli, abundant clear cytoplasm, and distinct cellular borders in hematoxylin and eosin staining. MGCT can be the first stage of malignancy in the development of testicular germ cell tumor (TGCT). Biopsies from men with maldescended testes have been reported to contain intratubular germ cell neoplasia, unclassified (IGCN) and MGCT in 1.8% of the examined cases (95% CI 0.5–4.6%). MGCT has also been found in testes of subfertile men and in the contralateral testis of patients with TGCT. MGCT is a frequent finding (19%) in the testicular tissue adjacent to an overt TGCT. Men with a high risk of TGCT may gain from screening for precursor lesions of TGCT with ultrasonography of the testes followed by a testicular biopsy if suspicious abnormalities are found: Treatment is high-voltage radiotherapy for intratubular germ cell neoplasia (IGCN), and orchidectomy for MGCT and germ cell tumor in situ, either intratubular seminoma or intratubular embryonal carcinoma. After local treatment, patients with precursor lesions can be followed with a surveillance program. The mRNA levels of invasion-related genes were evaluated based on a DNA microarray data set, and we found two gene abnormalities most relevant for the invasion of malignant germ cells: matrix metalloproteinase 9 (MMP9) and plasminogen activator, urokinase (PLAU) genes were up-regulated in a study comparing tissue samples of TGCT and IGCN.     

Intracranial germ cell tumor mimicking anorexia nervosa

Summary A previously healthy seventeen-year-old boy developed loss of weight, poor appetite, and aversion to food. Physical examination being normal, anorexia nervosa was suspected. Thirteen months later a CT scan revealed a mass in the third ventricle histologically proven to be a malignant teratoma. To our knowledge anorexia nervosa is only extremely rarely the presenting feature of intracranial germ cell tumors.Abbreviations CT computerized tomography - MRT magnetic resonance tomography - TR relaxation time - TE echo time - ms millisecond     

Development of tumor cell lines

A number of techniques are now in use in attempting to establish cell lines from human solid tumors. Although short-term cultures are readily attained, relatively few become established as permanent cell lines, especially from primary carcinomas where metastasis has not already occurred and from such organs as the breast, prostate, and pancreas. With recent advances in techniques and in our knowledge of growth factors, however, these difficulties may be resolved in the near future.     

Properties of human natural interferon-producing cells stimulated by tumor cell lines

Human peripheral blood mononuclear leukocytes cocultured with WISH human amnion cells or K562 tumor cells rapidly produce interferon-alpha (IFN). In the present report we characterize the IFN-producing cells (IPC) in this system by cell separation procedures, including panning with different monoclonal antibodies. Two types of cells which were responsible for the IFN production could be identified. The first IPC was classified as monocyte/macrophage because it was present in plastic-adherent cells and apparently carried the OKM1 antigenic marker. T and B lymphocytes were not involved in the IFN production. The second type of IPC was nylon wool-nonadherent, sheep erythrocyte rosette-negative, at least partly Fc receptor-negative and resided in light Percoll density gradient fractions. The natural killer activity and IFN-producing capacity was studied in such cells fractionated by the panning technique, utilizing HNK-1, OKT10 and OKM1 antibodies. When WISH and K562 were used as IFN inducers, HNK-1+ and OKT10+ cells with lytic activity against K562 produced little or no IFN. The IPC were instead confined to HNK-1- and OKT10- cells. With cells fractionated with respect to the OKM1 antigen, natural killer activity against K562 and WISH cells and IFN production stimulated by K562 cells resided in the OKM1+ cells. In marked contrast, cells producing IFN in response to WISH cells were OKM1-. The results therefore demonstrate a dissociation between natural killer cells against tumor cells and IPC stimulated by the same targets, suggesting that for at least certain targets/inducers they may represent distinct entities.     

Characterization of rearranged Y chromosomes in human testicular tumor cell lines

Cytogenetic analysis of four cell lines established from two different human testicular tumors revealed rearranged or missing Y chromosomes. Southern blot analysis and in situ hybridization with different Y-derived human DNA sequences revealed the existence of Y chromosomal material even in a line without a cytogenetically visible Y chromosome and clarified the composition of Y marker chromosomes.     

Intercellular karyotypic similarity in near-diploid cell lines of human tumor origins

Intercellular karyotypic compositions were studied in seven near-diploid cultured cell populations (including six cell lines) of human colorectal and fibrosarcoma origins. Within each population, 50–88% of cells had the same cell-line-specific karyotype. In five of these seven populations, coexisting minor cell types could readily be identified, implicating the frequent occurrence of mosaicism in transformed cell lines. Nearly 90% of these cell types showed a single chromosome loss or gain from the modal cell karyotype. Parameters that may assist in the evaluation of karyotypic diversity of a transformed cell line are discussed briefly.     

特异性hTERT启动子在肿瘤细胞中启动效率研究

目的证明人端粒酶亚单位（hTERT）启动子可以引导目的基因在肿瘤细胞中表达。方法先使用RT-PCR检测各细胞系中hTERT mRNA的表达,再使用PCR方法扩增hTERT启动子基因的不同长度片段,并构建重组质粒,使用BglⅡ和HindⅢ双酶切鉴定重组质粒并测序。最后使用Dual-Glo Luciferase Assay System激发荧光检测各细胞系中重组质粒中hTERT启动子的启动效率。结果 pGL3-204、pGL3-378、pGL3-1375重组质粒中的hTERT启动子序列与预期一致,质粒构建成功。hTERT启动子引导荧光基因的重组质粒在H460中启动效率最强可以达到SV40启动子的80%,而在FRO、U251中启动效率可以达到SV40启动子的30%左右。hTERT全长启动子序列的启动效率在ARO细胞系中比pGL3-204、pGL3-378中的启动子片段启动效率强,而在FRO、H460和U251细胞系中pGL3-204中的启动子片段启动效率最强。结论 hTERT启动子引导荧光基因的重组质粒,可以实现只在肿瘤细胞系中表达,但在正常细胞中不表达的效果。     

Bipolar (neural and myoblastic) phenotype in cell lines derived from human germ cell tumours of testis

Non-seminomatous germ cell tumours of the testis (NSGCT) form a heterogeneous group of neoplasms. Cell lines derived from NSGCT may provide useful data concerning the biology of neoplasic precursor germ cells, differentiation of tumour stem cells and the relationship between various tissue components of these tumours. Four NSGCT were studied, two mixed tumours composed of teratocarcinoma, yolk sac and trophoblastic elements, and two malignant teratomas with a massive neuroectodermal component, equivalent to primary neuroectodermal tumours (PNET) of the testis. The explanted tumours gave rise to various cell populations, including epitheloid cells, flattened large cells, spindle cells and tear drop cells of neuroblastic type. Ultrastructurally, cultured cells expressed various degrees of neural and muscular differentiation: neurosecretory granules, intermediate filaments of glial nature, and filaments resembling Z-bands. Cultured cells showed the expression of several neural and muscular markers, including neurofilaments, cytokeratin, actin, desmin, neuron-specific enolase, glial fibrillary acidic protein and HNK-1. In addition, three cases expressed HBA-71 antigen and two expressed MyoDI protein. All cases were aneuploid, and an isochromosome 12p, i(12p), was detected in three cases. Myoblastic and neural cells are the predominant tumour cells that grow in vitro, independent of the nature and composition of the primary germ cell tumour. A histogenetic relationship between germ cell tumours and small round cell tumours of childhood is suggested.     

Characterization of gains, losses, and regional amplification in testicular germ cell tumor cell lines by comparative genomic hybridization

We have performed comparative genomic hybridization on 12 testicular germ cell tumor (TGCT) cell lines and one paraffin-embedded surgical specimen to identify and characterize genome-wide gains and losses of chromosomes in these specimens. All specimens demonstrated overrepresentation of 12p. Other significant chromosomal gains, apart from 12p, included the X chromosome and chromosome arms 1q and 20q. Chromosomal losses were observed for chromosomes 4 and 18 and chromosome arms 2q, 9q, and 13q. Genomic differences were observed between an embryonal carcinoma component of a mixed tumor, 833K, and its cisplastin-resistant derivative line, 64CP, including losses of 6q23 approximately qter and 9p22 approximately q21. Five lines also demonstrated gain of 12p and additional 12p12 approximately p13 material. Similarly, two lines demonstrated gain of 12p and additional 12p11.2 approximately p12 material. The data supports the consistent gain of 12p in adult TGCT cell lines and additional regional amplification of 12p in some lines. This regional amplification has been observed in both primary tumor specimens and TGCT cell lines and may support a hypothesis that at least two different regions of 12p, one proximal and one distal, harbor genes important for the pathogenesis of testicular germ cell neoplasia.     

MAGE-1 and MAGE-3 tumor rejection antigens in human germ cell tumors.

The MAGE-1 and MAGE-3 genes are members of the melanoma antigen-encoding gene family. These genes encode for HLA-restricted tumor-associated rejection antigens recognized by cytotoxic T lymphocytes. MAGE-1 and MAGE-3 gene expression has been identified in a number of human malignancies, and MAGE-1 and MAGE-3 antigenic peptides are potential targets for tumor-specific immunotherapy. The only normal tissues known to express these genes are testicular germ cells and placental tissue. The objective of this study was to examine MAGE-1 and MAGE-3 antigens immunohistochemically in testicular germ cell tumors, including seminoma, a germ cell tumor frequently associated with a lymphoid infiltrate. Forty-three germ cell tumors (24 seminomas, six embryonal carcinomas and 13 mixed germ cell tumors), and 10 Leydig cell tumors were selected for study, and standard immunohistochemical techniques were used on formalin-fixed paraffin-embedded tissues using mouse monoclonal antibodies to MAGE-1 (clone M454) and MAGE-3 (clone 57B) antigens. MAGE-1 antigen was identified in 16.6% of seminomas. No embryonal carcinomas, yolk sac tumors, or teratomas contained MAGE-1 protein. MAGE-3 antigen was identified in 41.8% of seminomas, and this protein was not identified in embryonal carcinomas, yolk sac tumors, or teratoma. Spermatogonia and primary spermatocytes contained MAGE-1 and MAGE-3 antigen, and more mature forms, including spermatids, were weakly positive to negative. Leydig cell tumors were negative for MAGE-1 and MAGE-3. In seminoma, the presence of MAGE-1 and MAGE-3 antigens did not correlate with tumor size, tumor stage, the presence of a lymphoid infiltrate, or patient outcome. The low frequency of MAGE-specific HLA alleles in the population, the loss of the HLA class I antigens in neoplastic germ cells, and the finding that the majority of seminomas and all non-seminomatous germ cell tumors lacked MAGE-1 and MAGE-3 antigenic peptides indicate that immunotherapy directed towards MAGE-1 and MAGE-3 antigen is not a likely treatment option for seminoma and nonseminomatous germ cell tumors. The significance of MAGE-1 and MAGE-3 proteins in normal spermatogonia and primary spermatocytes will require additional study.     

Loss of heterozygosity of selected tumor suppressor genes in human testicular germ cell tumors

Human testicular germ cell tumors (TGCTs) are histologically heterogenous neoplasms with a variable malignant potential. Two main groups of germ cell tumors occur in men: seminomas and nonseminomas. In the present study, a set of four tumor suppressor genes was investigated in testicular cancers. CDH1, APC, p53, and nm23-H1 genes were tested for loss of heterozygosity (LOH). Thirty-eight testicular germ cell tumors (17 seminomas and 21 nonseminomas) were analyzed by PCR using restriction fragment length polymorphism or the dinucleotide/tetranucleotide repeat polymorphism method. An allelic loss of p53 at exon 4 was detected in five nonseminomas, whereas LOH of p53 at intron 6 occurred in one of the seminoma and two of the nonseminoma samples. Allelic losses of the APC gene were present in three seminomas and one nonseminoma, whereas one seminoma and three nonseminomas showed LOH of CDH1. The analysis of allelic losses showed no common structural genetic alterations in tumor tissues, although a different pattern of LOH was observed between the two main histological groups of TGCTs.     

Nonseminomatous germ cell tumor arising in splenogonadal fusion

Splenogonadal fusion is a rare congenital malformation in which the spleen is abnormally connected to the gonads or to the mesonephric derivatives. A few more than 150 cases have been described in the world literature. We report an additional case of splenogonadal fusion. A nonseminomatous germ cell tumor was found in the testicle involved in this splenogonadal fusion. To our knowledge, this is the third reported case of a testicular neoplasm associated with splenogonadal fusion and the first reported case of intra-abdominal nonseminomatous germ cell testicular tumor arising in this rare anomaly. The literature pertaining to splenogonadal fusion and the testicular tumor arising in this anomaly is briefly reviewed.     

Primary pulmonary alpha-fetoprotein-producing malignant germ cell tumor.

We are reporting the clinical and pathologic features of a primary, pulmonary, malignant germ cell tumor associated with a marked elevation of serum alpha-fetoprotein (38,427 ng/mL) and lactate dehydrogenase activity (756 U/L), in a 26-year-old female. This controversial, rare neoplasm has not been extensively discussed in the pathology literature. We emphasize the clinical importance of establishing this diagnosis in view of the favorable response to chemotherapy shown by malignant germ cell tumors.