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36 The role of karyotype instability in spontaneous neoplastic progression
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35 , THE RELATIONSHIP OF TUMORIGENICITY TO MULTIDRUG (MDR) RESISTANCE IN HUMANAND EXPERIMENTAL CANCERS. A.B. H i l l , W.T. Beck* and J.M. Trent, Radiation Oncology, Cancer Center, University of Arizona, Tucson, AZ 85724; *Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis TN 38101. We have evaluated whether CEM/VLBIOO cells which express the MDR phenotype via amplification of the P-glycoprotein gene retain their tumorigenicity. CEM/VLBIO0 has a 10fold amplification of the P-glycoprotein gene; the amplified genes are localized to an abnormal banding region (ABR). To assess the relationship ( i f any) of the acquisition of the MDR phenotype to tumorigenicity, mice were injected with 107 velban sensitive or resistant CEM cells. The results showed that 4/4 drug sensitive and 4/5 drug resistant cell lines formed tumors in nude mice within 5 or 6 weeks. Cells were then isolated from tumors and examined for P-glycoprotein gene amplification and tumor karyotype. None of the 4 sensitive tumors demonstrated amplification of P-glycoprotein (by Southern blot) or the presence of the ABR marker chromosome. In contrast, 3/4 drug resistant tumors contained both amplified P-glycoprotein genes and the ABR marker chromosome. These studies show conclusively that overexpression of Pglycoprotein does not a l t e r tumorigenicity in CEM leukemia cells. A.B.H. (Special Fellow) and J.M.T. (Scholar) are supported by the Leukemia Society of America.
3 6 *THE ROLE OF KARYOTYPE INSTABILITY IN SPONTANEOUS NEOPLASTIC PROGRESSION
L. S. Cram, M. F. Bartholdi, F. A. Ray, A. Brothman, and P. M. Kraemer, Experimental Pathology Group, Los Alamos National Laboratory, LOS Alamos, N.M. 87545 Karyotype instability in Chinese hamster cell cultures that spontaneously progress to become immortal, transformed, and finally tumorigenic was studied. The onset of karyotype instability was detected early in the sequence of events and accompanied immortalization. These cytogenetic and cellular changes preceeded in-vitro transformation and tumorigenicity. Karyotype analysis was performed by G-banding and high resolution flow karyotype analysis. Comparison of karyotypes of cultured cells and tumor derived cells indicated that karyotype instability continued in-vivo and that a variety of chromosomal changes accompanied tumor formation and progression. Although two recurring karyotype changes were prevalent (involving chromosomes #3 and #5) they did not correlate closely with tumor forming ability. These results will be contrasted with the partial and incomplete progression that occurred in Bloom syndrome fibroblasts. One strain of these cells had an extended life span and produced minute chromosomes after the cells were placed under conditions favoring the growth of transformed cells (1% serum). The application of flow karyotype analysis and new developments in the field will be described as they apply to this type of investigation. (Work performed under the auspices of the DOE and NIH, grant RR01315).
37 .
DIRECT SOLID TUMOR CYTOGENETICS USING THE TECHNIQUE OF PREMATURE CHROMOSOME CONDENSATION. Walter N. Hittelman, Phylisha Agbor, and Iskra Petkovic. University of Texas M. D. Anderson Hospital, Houston, Texas 77030. The purpose of this study was to determine the feasibility of using the technique of premature chromosome condensation in the cytogenetic analysis of solid tUmors. Cells obtained from dissociated solid tumors are fused with mitotic inducer tissue culture cells using Sendai virus as a fusogen resulting in the formation prematurely condensed chromosomes (PCC). The PCC from G1 and G2 tumor cells can then be G-, C-, or Q-banded and karyotyped in the same manner as mitotic chromosome preparations. Thus, there is no need to culture the tumor cells. To determine the origin of the fused cells from heterogeneous tumor specimen preparations, the fusion populations are treated with hypotonic and placed onto slides using a cytocentrifuge and processed for cell specific markers prior to acid-fixation in order to make a correlation between karyotype and cell type. We have begun to employ these techniques in the direct cytogenetic analysis of human lung tumor cells and cells obtained from normal, uninvolved lung tissue from the same patients. Our early results show that direct cytogenetic analysis can be performed on tumor and normal tissue cell suspensions using the PCC technique and that this approach should allow cytogenetic analysis of all tumor populations, irrespective of their proliferative status.
3 8 *HUMAN CYTOGENETIC ANALYSIS BY FLUORESCENCE IN SITU HYBRIDIZATION. D. Pinkel, J. Gray, B. Trask, J. Fuscoe, C. Collins, G. van den Engh, and H van Dekken. Lawrence Livermore National Laboratoryp Livermore, CA, USA. Cytogenetic analysis of solid tumors is complicated by the difficulty in producing bendable metaphase spreads whose karyotypes are representative of the important tumor cell populations. In situ hybridization using chromosome-specific nucleic acid probes permits distinct labeling of human chromosomes in poor metaphase spreads and interphase nuclei, which overcomes this limitation. Fluorescence detection of hybridization permits the simultaneous use of probes for several chromosomes and determination of the three dimensional positions of chromosomes in intact nuclei. Probes for repetitive sequences which which have the majority of their copies located in a small region of a single chromosome type are primarily useful for detection of aneuploldy. Translocation detection requires probe binding which spans a major portion of a chromosome. This can be accomplished using several repetitive probes or collections of unique sequences whose binding is distributed along the length of a chromosome. The exchange is then recognized by the presence of bound probe split between two chromosomes. An immediate application of fluorescence hybridization will be the analysis of characteristic aneuploidies in solid tumors. Eventually it may become possible to identify translocations in interphase nuclei, and to determine the role of chromosome arrangement in the formation and consequences of cytogenetic abnormalities.
35 , THE RELATIONSHIP OF TUMORIGENICITY TO MULTIDRUG (MDR) RESISTANCE IN HUMANAND EXPERIMENTAL CANCERS. A.B. H i l l , W.T. Beck* and J.M. Trent, Radiation Oncology, Cancer Center, University of Arizona, Tucson, AZ 85724; *Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis TN 38101. We have evaluated whether CEM/VLBIOO cells which express the MDR phenotype via amplification of the P-glycoprotein gene retain their tumorigenicity. CEM/VLBIO0 has a 10fold amplification of the P-glycoprotein gene; the amplified genes are localized to an abnormal banding region (ABR). To assess the relationship ( i f any) of the acquisition of the MDR phenotype to tumorigenicity, mice were injected with 107 velban sensitive or resistant CEM cells. The results showed that 4/4 drug sensitive and 4/5 drug resistant cell lines formed tumors in nude mice within 5 or 6 weeks. Cells were then isolated from tumors and examined for P-glycoprotein gene amplification and tumor karyotype. None of the 4 sensitive tumors demonstrated amplification of P-glycoprotein (by Southern blot) or the presence of the ABR marker chromosome. In contrast, 3/4 drug resistant tumors contained both amplified P-glycoprotein genes and the ABR marker chromosome. These studies show conclusively that overexpression of Pglycoprotein does not a l t e r tumorigenicity in CEM leukemia cells. A.B.H. (Special Fellow) and J.M.T. (Scholar) are supported by the Leukemia Society of America.
3 6 *THE ROLE OF KARYOTYPE INSTABILITY IN SPONTANEOUS NEOPLASTIC PROGRESSION
L. S. Cram, M. F. Bartholdi, F. A. Ray, A. Brothman, and P. M. Kraemer, Experimental Pathology Group, Los Alamos National Laboratory, LOS Alamos, N.M. 87545 Karyotype instability in Chinese hamster cell cultures that spontaneously progress to become immortal, transformed, and finally tumorigenic was studied. The onset of karyotype instability was detected early in the sequence of events and accompanied immortalization. These cytogenetic and cellular changes preceeded in-vitro transformation and tumorigenicity. Karyotype analysis was performed by G-banding and high resolution flow karyotype analysis. Comparison of karyotypes of cultured cells and tumor derived cells indicated that karyotype instability continued in-vivo and that a variety of chromosomal changes accompanied tumor formation and progression. Although two recurring karyotype changes were prevalent (involving chromosomes #3 and #5) they did not correlate closely with tumor forming ability. These results will be contrasted with the partial and incomplete progression that occurred in Bloom syndrome fibroblasts. One strain of these cells had an extended life span and produced minute chromosomes after the cells were placed under conditions favoring the growth of transformed cells (1% serum). The application of flow karyotype analysis and new developments in the field will be described as they apply to this type of investigation. (Work performed under the auspices of the DOE and NIH, grant RR01315).
37 .
DIRECT SOLID TUMOR CYTOGENETICS USING THE TECHNIQUE OF PREMATURE CHROMOSOME CONDENSATION. Walter N. Hittelman, Phylisha Agbor, and Iskra Petkovic. University of Texas M. D. Anderson Hospital, Houston, Texas 77030. The purpose of this study was to determine the feasibility of using the technique of premature chromosome condensation in the cytogenetic analysis of solid tUmors. Cells obtained from dissociated solid tumors are fused with mitotic inducer tissue culture cells using Sendai virus as a fusogen resulting in the formation prematurely condensed chromosomes (PCC). The PCC from G1 and G2 tumor cells can then be G-, C-, or Q-banded and karyotyped in the same manner as mitotic chromosome preparations. Thus, there is no need to culture the tumor cells. To determine the origin of the fused cells from heterogeneous tumor specimen preparations, the fusion populations are treated with hypotonic and placed onto slides using a cytocentrifuge and processed for cell specific markers prior to acid-fixation in order to make a correlation between karyotype and cell type. We have begun to employ these techniques in the direct cytogenetic analysis of human lung tumor cells and cells obtained from normal, uninvolved lung tissue from the same patients. Our early results show that direct cytogenetic analysis can be performed on tumor and normal tissue cell suspensions using the PCC technique and that this approach should allow cytogenetic analysis of all tumor populations, irrespective of their proliferative status.
3 8 *HUMAN CYTOGENETIC ANALYSIS BY FLUORESCENCE IN SITU HYBRIDIZATION. D. Pinkel, J. Gray, B. Trask, J. Fuscoe, C. Collins, G. van den Engh, and H van Dekken. Lawrence Livermore National Laboratoryp Livermore, CA, USA. Cytogenetic analysis of solid tumors is complicated by the difficulty in producing bendable metaphase spreads whose karyotypes are representative of the important tumor cell populations. In situ hybridization using chromosome-specific nucleic acid probes permits distinct labeling of human chromosomes in poor metaphase spreads and interphase nuclei, which overcomes this limitation. Fluorescence detection of hybridization permits the simultaneous use of probes for several chromosomes and determination of the three dimensional positions of chromosomes in intact nuclei. Probes for repetitive sequences which which have the majority of their copies located in a small region of a single chromosome type are primarily useful for detection of aneuploldy. Translocation detection requires probe binding which spans a major portion of a chromosome. This can be accomplished using several repetitive probes or collections of unique sequences whose binding is distributed along the length of a chromosome. The exchange is then recognized by the presence of bound probe split between two chromosomes. An immediate application of fluorescence hybridization will be the analysis of characteristic aneuploidies in solid tumors. Eventually it may become possible to identify translocations in interphase nuclei, and to determine the role of chromosome arrangement in the formation and consequences of cytogenetic abnormalities.