An improved explant cell culture technique to avoid selection of prostatic adenocarcinoma cells toward diploid cells is described. This method is based on 1) histologically characterized tissue ...explants, 2) the use of polyethylenteraphthalate (PET) membranes as growth surface, which are part of special inserts in six-well-plates to allow 3) cocultivation with heterologous fibroblasts, and 4) coating of the membranes with elements of the extracellular matrix. The main characteristic of this particular approach is the serial transfer of the tissue explant from one membrane to the other. Up to ten serial transfer steps could be performed to produce cell monolayers growing out of the same tissue specimen. Using this approach, 21 prostatic carcinoma specimens that were obtained from 13 primary prostatic adenocarcinomas after radical prostatectomy were cultivated. Ploidy of the cells was monitored by fluorescence in situ DNA hybridization using the centromere specific DNA probes pUC1.77, pα7t1, and pY3.4. Interestingly, a high aneuploidy rate of the cell cultures was found with maintainance of aneuploidy in 18 (86%) of the 21 paraffin-embedded cancer tissue specimens with proved aneuploidy. Although a slight decrease of the proportion of aneuploid cells during serial transfer was observed, significant aneuploid cell populations were retained up to a maximum of ten transfer steps. These findings indicate that selection toward diploid cells can be prevented by improved cell culture techniques that mimic the in vivo situation.
To investigate the potential genetic changes underlying the progression of human hormone-resistant prostate cancer, we related chromosomal alterations of the DU 145 cell line and a subline isolated ...form a metastasis in an orthotopic model to tumorigenicity, metastasis and chemoresistance. In 15 mice 1 × 10
5 DU 145 cells were injected into the dorsal prostate. From a resulting paraaortic lymphnode metastasis, we isolated a subline (DU 145 MN1), which was injected into 15 nude mice. The sulforhodamine B (SRB) assay was used to analyze cell doubling time and the IC
50 of cisplatin and 5-fluorouracil for both cell lines. Cytogenetic characterization was performed with conventional karyotype analysis and fluorescence in situ hybridization (FISH). After orthotopic implantation of DU 145 cells tumorigenicity was 100% whereas only 2 mice revealed lymphnode metastases. In contrast, the take rate after implantation of DU 145 MN1 was 100%, with lymphnode metastases in 7 mice. The SRB assay revealed a 8-fold increased IC
50 for cisplatin and a 2.5-fold increase for 5-FU in DU 145 MN1 as compared to DU 145 cells. There was gain of a chromosome 8 and only two copies of chromosome 17 in the DU 145 MN1 cells as compared to the parental cell line. The emergence of an i(9)(q10) in addition to two normal chromosome 9 homologues in the DU 145 MN1 cell line was confirmed by FISH using a chromosome 9-specific painting probe. In summary, clonal evolution of the chromosomal changes following repeated orthotopic implantation, may assist in locating the genes involved in the progression and chemoresistance of human hormone-resistant prostate cancer.
Because cytogenetic analysis and fluorescence in situ hybridization (FISH) techniques fail to provide information about the immunological phenotype with respect to differentiation and malignancy of ...in vitro growing cells, we developed a system which combines immunophenotyping and FISH. Using immunological detection of the proliferation fraction in short term human cell cultures from different origins by Ki-67 labeling and genetic characterization by FISH, both signals were detectable simultaneously on the same monolayer. Because determination of the proliferation behavior of tumor cells by Ki-67 labeling has now become a widespread laboratory tool, this particular technique may be very useful in tumor cell characterization. This approach allows genetic changes to be assigned to a particular immunological phenotype of individual cells.
Clonal chromosomal changes were detected in three of five sporadic angiomyolipomas of the kidney irrespective of a solitary or multifocal appearance of this benign tumor type. No specific chromosomal ...changes have been identified. Including the cytogenetic data of the four renal angiomyolipomas reported in the literature, trisomy 7 as the single clonal chromosomal abnormality was detected in two angiomyolipomas. Because trisomy 7 has been reported in both neoplastic and nonneoplastic kidney cells, it may be assumed that trisomy 7 is already physiologically resident in renal cells but undergoes positive selection in this tumor type.
In very rare patients tetrasomy of chromosome 8 is observed as the only karyotypic event. We present a 74-year-old man with acute monocytic leukemia (FAB M5) displaying 46,XY/48,XY, +8, +8 in ...unstimulated bone marrow cells. The patient died 2 weeks after diagnosis. Tetrasomy 8 appears to identify a subgroup of acute nonlymphocytic leukemia. It may indicate a poor prognosis.
Nonradioactive in situ hybridization provides a rapid method for detecting specific nucleic acid sequences. In this study of a patient with acute leukemia, we applied in situ hybridization for ...identification of a marker chromosome and determination of the number of copies of this marker in interphase nuclei using a biotinylated Yq-specific DNA probe (pY3.4). We show that nonradioactive interphase in situ hybridization can be a useful method for karyotypic analysis in addition to routine cytogenetic techniques in neoplastic disorders.
We report on a recurrent intracranial hemangiopericytoma cytogenetically studied after short-term culture. The tumor had a uniform karyotype 47,XX,add(7)(q21),t(12;19)(q13;q13.3),del(13)(q14q22), ...+21. Remarkably, one case with an identical reciprocal (12;19) translocation has been previously reported as the sole cytogenetic change in a recurrent retroperitoneal hemangiopericytoma. This nonrandom structural change may characterize a subentity of hemangiopericytoma and might be of diagnostic value.
Nine endometrial carcinomas were examined for numerical aberrations of the chromosomes 1, 7 and X by fluorescence in situ hybridization using highly repetitive chromosome-specific probes. In ...addition, a combination of a centromeric and a telomeric chromosome 1 probe was applied to detect structural chromosome 1 aberrations. Chromosome aberrations were found in six tumors. In four of these, an imbalance between 1q12 and 1p36 was detected, indicating the presence of an extra 1p− chromosome. In regard to the chromosomes 7 and X, monosomies and trisomies were found. Intratumoral genetic heterogeneity in endometrial carcinomas was detectable by FISH and flow cytometry. In conclusion, our findings confirm that chromosome 1 is frequently involved in structural chromosome changes, indicating chromosome 1 to be of importance in the evolution of endometrial carcinoma.
In a previously studied family with inherited renal cell carcinoma (RCC), RCC was shown to segregate with a constitutional balanced t(3;8)(p14.2;q24.1). In addition, we recently showed that in a RCC ...tumor from this family the constitutional translocation became unbalanced, suggesting a genetic mechanism that may be associated with the primary genetic events of tumorigenesis. We now report that the RCC tumor cells from this case showed additional cytogenetic alterations, possibly related to tumor progression, which include an additional tumor-specific translocation involving band 14 of chromosome 13. Because this band contains the retinoblastoma (RB) gene, we examined the tumor for aberrations in the RB gene using DNA sequence polymorphism analysis and pulsed-field gel electrophoresis (PFGE), but did not detect alterations in the RB gene.
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