Molecular pathology: retrospective archival analyses.
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DNA↗
Biomedical subjects
M A Israel
Publications and source records attributed to M A Israel.
The expression of multiple proto-oncogenes is differentially regulated during retinoic acid induced maturation of human neuroblastoma cell lines.
Human neuroblastoma (NB) is a highly malignant tumor arising in cells that originate in the embryonal neural crest. Several lines of investigation suggest that both NB and other tumors of developing tissues are blocked in their ability to differentiate and achieve growth arrest. Since in vivo differentiation of NB has been frequently observed and may be of clinical importance (Fox et al., 1959; Evans et al., 1976), we have utilized the in vitro induction of NB differentiation by retinoic acid (RA) to study the molecular events associated with NB differentiation. We have focused our studies on changes that occur in the expression of various proto-oncogenes during NB tumors cell differentiation because proto-oncogenes are likely to be of central importance in mediating processes critical for cellular growth and maturation. In these studies, we have found that the expression of no fewer than five proto-oncogenes including c-Ha-ras, c-ets-1, and c-fos change during the differentiation of NB cells, while the expression of c-erb-B changes in association with the arrest of growth that occurs during NB differentiation. In some cases the altered expression of a proto-oncogene was transcriptionally regulated, while in others post-transcriptional mechanisms were important.
Cell Differentiation↗
Developmentally regulated genes in neuroblastoma.
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Cell Line↗
Isolation of amplified and overexpressed DNA sequences from adriamycin-resistant human breast cancer cells.
An MCF-7 human breast cancer cell line was selected which was 200-fold more resistant to Adriamycin than the wild type cell line. This Adriamycin-resistant (AdrR) cell line exhibited a multidrug-resistant phenotype and was cross-resistant to a wide range of antineoplastic agents including Vinca alkaloids, anthracyclines, and epipodophyllotoxins. Cytogenetic analysis of the AdrR cell line showed the presence of homogeneously staining regions on several chromosomes which were not present in the parental cell line. Using the technique of in-gel renaturation, DNA sequences which were amplified 50- to 100-fold in the AdrR cell line and which covered a total of over 140 kilobases were isolated. In addition, AdrR cells were found to contain amplified and overexpressed sequences which were homologous to hamster P-glycoprotein gene sequences. A hamster cDNA P-glycoprotein gene probe was used to screen a lambda gt10 cDNA library made from human AdrR cell line mRNA and human cDNA sequences homologous to the P-glycoprotein gene were isolated. Hybridization studies with the cloned human cDNA (pADR1) showed that the AdrR MCF-7 cell line contained a 60-fold amplification of this DNA sequence and that polyadenylated mRNA from the AdrR cell line contained a 4.8-kilobase transcript which was overexpressed 45-fold. There was a direct correlation between DNA and RNA copy number of this sequence and level of resistance among several MCF-7 Adriamycin-resistant cell lines. In situ hybridization studies demonstrated that the human P-glycoprotein gene sequence was found on chromosome 7q21.1 in normal human lymphocytes and that amplified DNA sequences isolated from the AdrR MCF-7 cells by the in-gel hybridization technique were linked to the human P-glycoprotein sequences in the homogeneously staining regions in the AdrR cells.
Base Sequence↗
Translocation of c-sis protooncogene in peripheral neuroepithelioma.
Molecular genetic analysis of the c-sis protooncogene was performed on two neuroepithelioma cell lines carrying a t(11;22)(q24;q12). The c-sis protooncogene was found by in situ hybridization to be translocated from its germline position on chromosome #22 to the derivative chromosome #11 in each cell line. However, it was not rearranged or amplified in either cell line examined. In addition, we did not detect c-sis transcripts in Northern blots of poly (A)+ RNA. This is similar to results found in Ewing's sarcoma, which carries a cytogenetically indistinguishable translocation from neuroepithelioma.
Adolescent↗
Molecular markers of neuroendocrine development and evidence of environmental regulation.
We constructed a human pheochromocytoma cDNA library and used differential hybridization to human pheochromocytoma and human neuroblastoma cDNA probes to isolate genes that are highly expressed in the adrenal medullary neuroendocrine tumor, pheochromocytoma, but not in the more immature embryonal tumor of adrenal medulla, neuroblastoma. Two cDNA clones, pG8 and pG2, were more highly expressed in normal and neoplastic chromaffin tissue than they are in neuroblastoma. Furthermore, they are expressed in a remarkably limited number of other human tumors or normal tissues. pG8 is highly expressed in medullary thyroid carcinoma, another tumor of neural crest origin, which can occur in association with pheochromocytoma in the multiple endocrine neoplasia type II syndrome. pG2 is highly expressed in the adrenal cortex, an endocrine gland thought to be embryologically unrelated to the neural crest-derived adrenal medulla. The expression of both pG8 and pG2 can be induced in human neuroblastoma cells with dexamethasone, suggesting a mechanism by which glucocorticoids may influence development of a neuroendocrine phenotype.
Adrenal Gland Neoplasms↗
Differential protooncogene expression characterizes histopathologically indistinguishable tumors of the peripheral nervous system.
We have found highly predictable patterns of protooncogene expression in cell lines and tumor tissue of neuroblastoma (NB), a tumor of the peripheral nervous system (PNS). These patterns make it possible to recognize two different genetically definable subgroups among histopathologically indistinguishable tumors. Additionally, we have identified a difference in neurotransmitter biosynthetic enzyme activity in these two subgroups of NB. The patterns of protooncogene expression and neurotransmitter biosynthetic enzymes suggests that these tumors arise in different cells of the PNS.
Cell Line↗
Patterns of proto-oncogene expression: a novel approach to the development of tumor markers.
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Animals↗
Genetic abnormalities as biological tumor markers.
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Aneuploidy↗
Analysis of pp60c-src protein kinase activity in human tumor cell lines and tissues.
We have evaluated the level of pp60c-src protein kinase activity in a variety of human tumor tissues and human tumor cell lines, and have estimated the abundance of the c-src protein in several of these tissues and cell lines. All cell lines derived from tumors of neuroectodermal origin that express a neural phenotype were found to possess c-src molecules with high levels of tyrosine-specific protein kinase activity. In contrast, cell lines derived from tumors of neuroectodermal origin that do not express neural characteristics, such as glioblastomas and melanomas, were found to have pp60c-src molecules with low levels of protein kinase activity. A similar pattern was observed when we analyzed the activity of c-src molecules extracted directly from corresponding tumor tissues. Analysis of human tumor cell lines derived from tissues other than those of neuroectodermal origin revealed that pp60c-src protein kinase activity was low in most cases. Exceptions to this observation were all rhabdomyosarcoma, osteogenic sarcoma, Ewing's sarcoma, and colon carcinoma lines tested. Comparison of pp60c-src kinase activity in normal skeletal muscle and rhabdomyosarcoma tissue and in normal breast tissue and breast adenocarcinoma tissue revealed that pp60c-src kinase activity was specifically elevated in the tumor tissues in both cases. However, the amount of pp60c-src protein in both normal and tumor tissues was found to be similar. These observations suggest that increases in the specific activity of the pp60c-src phosphotransferase in some rhabdomyosarcomas and breast carcinomas may be a characteristic acquired during the malignant transformation of the cells that is retained in cell lines established from these tumors.
Cell Line↗
Cytogenetic characterization of selected small round cell tumors of childhood.
Small, round, blue-cell tumors (SRCT), including rhabdomyosarcoma, Ewing's sarcoma of bone and soft tissue, mesenchymal chondrosarcoma, small cell osteosarcoma, hemangiopericytoma, neuroblastoma, peripheral neurectodermal tumor (peripheral neuroepithelioma of bone and soft tissue), and the malignant small cell tumor of the thoracopulmonary region described by Askin (Askin's tumor), are often difficult to distinguish by light microscopy. We have evaluated the cytogenetics of these tumors by studying 24 tumor explants in short-term culture and 22 tumor cell lines. In Ewing's sarcoma (a tumor of unknown histogenesis), and in peripheral neuroepithelioma and Askin's tumor (tumors with evidence of neural origin), we have observed an indistinguishable t(11;22) translocation.
Adolescent↗
Translocation breakpoint mapping: molecular and cytogenetic studies of chromosome 22.
A number of clinically significant human diseases are associated with rearrangements of chromosome #22. These include the t(9;22) of chronic myelogenous leukemia (CML), the t(8;22) of Burkitt's lymphoma (BL), the t(11;22)(q23;q11) constitutional rearrangement and the t(11;22) of Ewing's sarcoma (ES) and neuroepithelioma (NE). All of these translocations have breakpoints in 22q11. Using a molecular cytogenetic approach and various cloned portions of the lambda light chain gene as probe, we have assigned a linear order within 22q11 to these breakpoints. Using chromosomal in situ hybridization we have determined that the 22q11 breakpoint in BL2, a t(8;22) Burkitt's lymphoma is proximal to the breakpoint of the t(9;22) of CML. We have demonstrated that the 22q11 breakpoint of PA682, another t(8;22) BL cell line, interrupts the lambda light chain locus. Using a combination of variable and constant region probes and PA682 cells, we have shown that the lambda light chain locus is oriented such that V lambda is proximal to C lambda in 22q11. Our results for the constitutional t(11;22) indicate that the 22q11 breakpoint is distal to that of BL, proximal both to that of CML and ES and, in addition, it interrupts the C lambda gene cluster. Our studies of the 22q11 breakpoint of the t(11;22) of ES and NE suggest that they are the most distal of the breakpoints we studied on chromosome #22.
Burkitt Lymphoma↗
Comparison of constitutional and tumor-associated 11;22 translocations: nonidentical breakpoints on chromosomes 11 and 22.
Recurring, site-specific chromosomal rearrangements are associated with several human syndromes and malignant disorders. Such nonrandom translocations involving chromosome 22 in band q11 are numerous and found to be associated with a diversity of neoplasms as well as constitutional disorders. Chromosome 11 in bands q23-q24 is similarly involved in several types of tumors as well as in a recurring constitutional reciprocal translocation with chromosome 22. Here we report the use of chromosomal in situ hybridization to compare the translocation breakpoints in the cytologically indistinguishable constitutional t(11;22) and the tumor-related t(11;22) associated with Ewing sarcoma and peripheral neuroepithelioma. We have shown that the breakpoints can be distinguished from each other with respect to the locus encoding the constant region of the Ig lambda light chain (C lambda) at 22q11 and the ETS1 locus at 11q23----q24; ETS1 has been called hu-ets-1 or human c-ets-1. The tumor-associated chromosome 11 breakpoint is also different from those of leukemias with t(9;11) and t(4;11) translocations. Southern-blot analysis showed no rearrangement of ETS1 in these disorders in the region detected by our probe. ETS1 has also been mapped more precisely to 11q23.3----q24 by in situ hybridization to cells from an individual with an 11q23.3----qter deletion.
Cell Line↗
Analysis of pp60c-src protein kinase activity in hamster embryo cells transformed by simian virus 40, human adenoviruses, and bovine papillomavirus 1.
We have examined the effect of DNA tumor virus transformation of primary hamster embryo cells on the tyrosyl kinase activity of pp60c-src. Our present study demonstrates that some clones of hamster embryo cells transformed by simian virus 40, adenovirus type 2, adenovirus type 12, or bovine papillomavirus 1 can possess elevated pp60c-src kinase activity when compared with normal hamster embryo cells. However, other clones of hamster embryo cells transformed by these same viruses were found to have normal levels of pp60c-src kinase activity. In those clones of transformed cells where pp60c-src kinase activity was elevated, the increased levels of kinase activity were the result of an apparent increase in the specific activity of the pp60c-src phosphotransferase rather than an increase in the amount of the src gene product. Additionally, pp60c-src was not found to be physically associated with tumor antigens known to be encoded by these viruses. These results indicate that elevated levels of pp60c-src kinase activity can be found in hamster embryo cells transformed by several different DNA tumor viruses and suggest that the molecular mechanism by which pp60c-src kinase activity is elevated may differ from that previously observed in polyomavirus-transformed cells. These results also imply that elevation of pp60c-src kinase activity is not required for the transformation of hamster cells by these viruses.
Adenoviruses, Human↗
A subclass of polyomavirus middle tumor antigen binds to DNA cellulose.
We examined the binding of polyomavirus large (L-T)-, middle (M-T)-, and small-tumor antigens to DNA cellulose. At pH 6.0, the majority of L-T bound to calf thymus DNA cellulose, while little or no small tumor antigen was retained under these conditions. Unexpectedly, a small but reproducible proportion of M-T bound to both native and denatured DNA cellulose. M-T encoded by polyomavirus mutant dl 8, which expressed shortened L-T and M-T, bound to DNA, indicating that the deleted sequences are not required for DNA binding. Also, M-T from transformed BMT-1 rat cells, which synthesize exclusively this polyomavirus tumor antigen, bound to DNA, indicating that its binding is not due to association with other polyomavirus-encoded proteins. Using the DNA fragment immunoassay, we found that, under conditions in which L-T bound specifically to DNA fragments containing viral regulatory sequences, no viral DNA fragments were bound by M-T. The existence of distinct subpopulations of M-T that differ in their DNA-binding properties was indicated by rebinding experiments in which M-T that had bound to DNA cellulose rebound very efficiently, while that which had not been originally retained by DNA cellulose rebound poorly. Furthermore, the M-T-pp60 c-src complex did not bind to DNA cellulose. These data suggest that polyomavirus M-T is heterogeneous, consisting of populations of molecules that differ in their interactions with DNA cellulose.
Antigens, Viral, Tumor↗
The evolution of clinical molecular genetics. Neuroblastoma as a model tumor.
The use of cytogenetics and molecular genetics to elucidate mechanisms underlying the pathogenesis of human cancer has led to important advances in our understanding of cancer etiology. The identification of a reciprocal (11;22) translocation in peripheral neuroectodermal tumors and N-myc amplification in classical childhood neuroblastoma provides new guidelines for the diagnostic evaluation of peripheral nervous system tumors. New approaches to the staging of these tumors are likely to include the evaluation of genetic alterations associated with tumor progression. Also, new therapeutic approaches will be focused on the modification of specific molecular targets responsible for neoplastic growth.
Cell Line↗
Increased N-myc expression following progressive growth of human neuroblastoma.
Many human neuroblastoma tumors and cells in culture contain amplified N-myc DNA and this is associated with tumor stage. We have analyzed pairs of neuroblastoma cell cultures derived from two patients at the time of diagnosis and after tumor progression following the initiation of therapy. Cell cultures derived after progression have increased expression of N-myc RNA. In one pair this increase is associated with increased N-myc DNA amplification; in the other, amplification decreases and activation of N-myc is most likely the result of a regulatory change. Analysis of the pattern of DNA amplification in these cell cultures demonstrates additional changes that might be associated with tumor progression.
Base Sequence↗
Stimulation of pp60c-src tyrosyl kinase activity in polyoma virus-infected mouse cells is closely associated with polyoma middle tumor antigen synthesis.
We have examined the effect of polyoma virus infection of primary mouse embryo cells on the tyrosyl kinase activity associated with the cellular src gene product, pp60c-src. The results of our studies demonstrate that infection of mouse cells with wild-type polyoma virus or viral mutants capable of transforming rodent cells in culture and inducing tumors in animals results in the stimulation of pp60c-src tyrosyl kinase activity. The level of pp60c-src kinase stimulation in infected cells was found to be proportional to both the oncogenic potential of the virus strain used for infection and the characteristic phenotype of rodent cells transformed by the various strains of polyoma virus. Stimulation of pp60c-src kinase activity was not observed in mouse cells infected with transformation-defective strains of polyoma virus. In examining the kinetics of pp60c-src kinase stimulation in mouse cells at various times following wild-type polyoma virus infection, we found that the level of pp60c-src kinase activity correlated directly with the synthesis of polyoma virus-encoded tumor antigens. By comparing wild-type polyoma virus with other viral mutants in these experiments, we conclude that the stimulation of pp60c-src kinase activity in mouse cells following polyoma virus infection is associated with the synthesis of middle tumor antigen.
Animals↗