Biomedical subjects
A M Killary
Publications and source records attributed to A M Killary.
A tumor suppressor locus within 3p14-p12 mediates rapid cell death of renal cell carcinoma in vivo.
High frequency loss of alleles and cytogenetic aberrations on the short arm of chromosome 3 have been documented in renal cell carcinoma (RCC). Potentially, three distinct regions on 3p could encode tumor suppressor genes involved in the genesis of this cancer. We report that the introduction of a centric fragment of 3p, encompassing 3p14-q11, into a highly malignant RCC cell line resulted in a dramatic suppression of tumor growth in athymic nude mice. Another defined deletion hybrid contained the region 3p12-q24 of the introduced human chromosome and failed to suppress tumorigenicity. These data functionally define a tumor suppressor locus, nonpapillary renal carcinoma-1 (NRC-1), within 3p14-p12, the most proximal region of high frequency allele loss in sporadic RCC as well as the region containing the translocation breakpoint in familial RCC. Furthermore, we provide functional evidence that NRC-1 controls the growth of RCC cells by inducing rapid cell death in vivo.
Analysis of the functional role of chromosome 10 loss in human glioblastomas.
Molecular and cytogenetic analyses of primary brain tumors have shown that losses on chromosome 10 occur very frequently in human glioblastoma multiforme suggesting the presence of a glioma-associated tumor suppressor gene on this chromosome. To examine this hypothesis, a copy of chromosome 10 derived from a human fibroblast cell line was introduced into the human glioma cell line U251 by microcell-mediated chromosomal transfer. A human chromosome 2 was also independently introduced into U251 cells. The presence of novel chromosomes or chromosomal fragments was confirmed by molecular and karyotypic analyses. The hybrid clones containing a transferred chromosome 10 exhibited a suppression of their transformed and tumorigenic phenotype in vivo and in vitro, whereas cells containing a transferred chromosome 2 failed to alter their phenotype. The hybrid cells containing a transferred chromosome 10 displayed a significant decrease in their saturation density and an altered cellular morphology at high cell density but only a slight decrease in their exponential growth rate. A dramatic decrease was observed in the ability of cells with an introduced chromosome 10 to grow in soft agarose. The introduction of chromosome 10 completely suppressed tumor formation when the hybrid cells were injected into nude mice. These findings indicate that chromosome 10 harbors a tumor suppressor gene that is directly involved in glioma oncogenesis.
A homozygous deletion on chromosome 3 in a small cell lung cancer cell line correlates with a region of tumor suppressor activity.
Small cell lung cancer (SCLC) tumors frequently display deletions on the short arm of chromosome 3 suggesting the existence of a 'tumor suppressor' gene within that region whose functional inactivation may be involved in tumorigenesis. Recently, a hybrid, HA(3)BB9F, was identified that contains a small fragment of human chromosome 3 of approximately 2 Mb on a mouse (A9) background (Killary et. al., 1992). This hybrid was utilized to define a functional tumor suppressor gene within 3p22-p21 which could suppress the tumorigenic properties of the mouse fibrosarcoma cell line. The existence of a tumor suppressor gene in the region 3p22-p21 is supported by the present report which describes the assessment of 89 SCLC and 32 non-SCLC lung cancer tumors and cell lines for the existence of a homozygous deletion(s) at 43 loci on the short arm of chromosome 3. One of the SCLC cell lines was found to harbor a homozygous deletion involving the loss of five markers on chromosome 3p. All five of the markers map to the region 3p21.3-p21.2 and four of the five markers are located within the chromosome 3 fragment exhibiting properties of tumor suppression in the HA(3)BB9F hybrid. The other tumors analysed all retained at least one copy of each of the markers assessed.
Definition of a tumor suppressor locus within human chromosome 3p21-p22.
Cytogenetic abnormalities and high-frequency allele losses involving the short arm of human chromosome 3 have been identified in a variety of histologically different neoplasms. These findings suggest that a tumor-suppressor gene or genes may be located in the region of 3p14-p25, although there has been no definitive functional proof for the involvement of a particular region of 3p. We report a rapid genetic assay system that has allowed functional analysis of defined regions of 3p in the suppression of tumorigenicity in vivo. Interspecific microcell hybrids containing fragments of chromosome 3p were constructed and screened for tumorigenicity in athymic nude mice. Hybrid clones were obtained that showed a dramatic tumor suppression and contained a 2-megabase fragment of human chromosomal material encompassing the region 3p21 near the interface with 3p22. With these hybrid clones, we have defined a genetic locus at 3p21-p22 intimately involved in tumor suppression.
Construction of a human chromosome 4 YAC pool and analysis of artificial chromosome stability.
In order to construct a human chromosome 4-specific YAC library, we have utilized pYAC4 and a mouse/human hybrid cell line HA(4)A in which the only human chromosome present is chromosome 4. From this cell line, approximately 8Mb of chromosome 4 have been cloned. The library includes 65 human-specific clones that range in size from 30kb to 290kb, the average size being 108kb. In order to optimize the manipulation of YAC libraries, we have begun to investigate the stability of YACs containing human DNA in yeast cells; these studies will also determine if there are intrinsic differences in the properties of chromosomes containing higher eukaryotic DNAs. We are examining two kinds of stability: 1] mitotic stability, the ability of the YAC to replicate and segregate properly during mitosis, and 2] structural stability, the tendency of the YAC to rearrange. We have found that the majority of YACs examined are one to two orders of magnitude less stable than authentic yeast chromosomes. Interestingly, the largest YAC analyzed displayed a loss rate typical for natural yeast chromosomes. Our results also suggest that increasing the length of an artificial chromosome improves its mitotic stability. One YAC that showed a very high frequency of rearrangement by mitotic recombination proved to be a mouse/human chimera. In contrast to studies using total human DNA, the frequency of chimeras (i.e., mouse/human) in the YAC pool appeared to be low.
Dissociation of cytotoxicity and DNA cleavage activity induced by topoisomerase II-reactive intercalating agents in hamster-human somatic cell hybrids.
Previous studies using the mutant Chinese hamster ovary cell line VpmR-5 indicate that its resistance to epipodophyllotoxins and intercalating agents is likely to be mediated through a qualitative change in type II topoisomerase that confers resistance to drug-stimulated DNA cleavage activity. In a further investigation of the genetic basis of drug resistance in VpmR-5 cells, we fused a hypoxanthine-guanine phosphoribosyl transferase-deficient subline of VpmR-5 (Vtgm-6) with normal human lymphocytes and analyzed the resultant hybrid lines (HL) for altered drug sensitivity. In all, 3 of 16 hybrid clones exhibited partial reconstitution of sensitivity to etoposide, mitoxantrone, doxorubicin, and 5-iminodaunorubicin while retaining complete resistance to m-AMSA. However, enhanced sensitivity to drug-induced DNA cleavage activity was observed only for etoposide. Biochemical and molecular-marker analysis of the hybrids failed to identify human chromosome 17 (the provisional location of TOP2) or any other human chromosome that is consistently and uniquely associated with drug sensitivity. We therefore sought to verify the chromosomal assignment of TOP2 by Southern blot hybridization of TOP2 cDNA on a human hybrid mapping panel and confirmed its location on chromosome 17. However, no hybridizing sequence to the TOP2 cDNA was found in any of the 16 Vtgm-6 hybrid lines. Efforts to select more directly for human chromosome 17 VpmR-5 hybrids using microcell fusion of mouse A9 cells carrying human 17 linked to pSV2neo were unsuccessful. None of the five hybrid clones thus obtained had 17q markers, including the gene for TOP2. Although the mechanism underlying partial reversion to a drug-sensitive phenotype in the original Vtgm-6 hybrid lines has yet to be defined, the data obtained in these lines indicate that anthracycline- and anthracenedione-induced cytotoxic effects can be dissociated from DNA cleavage activity. This suggests that pathways distal to cleavable-complex formation or, alternatively, independent of interactions with topoisomerase II that involve other intracellular targets are important in mediating the cytotoxicity produced by these drugs.
Genetic analysis of indefinite division in human cells: evidence for a cell senescence-related gene(s) on human chromosome 4.
Earlier studies had demonstrated that fusion of normal with immortal human cells yielded hybrids having limited division potential. This indicated that the phenotype of limited proliferation (cellular senescence) is dominant and that immortal cells result from recessive changes in normal growth-regulatory genes. In additional studies, we exploited the fact that the immortal phenotype is recessive and, by fusing various immortal human cell lines with each other, identified four complementation groups for indefinite division. Assignment of cell lines to specific groups allowed us to take a focused approach to identify the chromosomes and genes involved in growth regulation that have been modified in immortal cells. We report here that introduction of a normal human chromosome 4 into three immortal cell lines (HeLa, J82, T98G) assigned to complementation group B resulted in loss of proliferation and reversal of the immortal phenotype. No effect on the proliferation potential of cell lines representative of the other complementation groups was observed. This result suggests that a gene(s) involved in cellular senescence and normal growth regulation resides on chromosome 4.
Chromosomal localization of the human annexin III (ANX3) gene.
The annexins or lipocortins are a new family of calcium-dependent phospholipid-binding proteins. Annexin III has been previously identified as inositol 1,2-cyclic phosphate 2-phosphohydrolase (EC 3.1.4.36), an enzyme of inositol phosphate metabolism, and also as placental anticoagulant protein III, lipocortin III, calcimedin 35-alpha, and an abundant neutrophil cytoplasmic protein. In this study, the gene (ANX3) encoding annexin III was localized to human chromosome 4 at band q21 (q13-q22) by (1) polymerase chain reaction analysis of a human-rodent hybrid cell panel, confirmed by genomic Southern blot analysis of the same panel with a cDNA probe and (2) in situ hybridization with a cDNA probe.
Dinucleotide repeat polymorphism at the D3S196 locus.
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Dinucleotide repeat polymorphism at the D4S174 locus.
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Assignment of retinoblastoma susceptibility gene to mouse chromosome 14.
A human cDNA probe was used to screen a panel of mouse-Chinese hamster somatic cell hybrids to determine the chromosomal location of the retinoblastoma susceptibility gene (Rb-1) in mouse. The Rb-1 gene mapped to mouse chromosome 14. Thus, the retinoblastoma susceptibility gene is syntenic with esterase 10 (the mouse homolog of human esterase D). The chromosomal assignment of the mouse Rb-1 gene was further confirmed by using the same probe to study mouse-rat microcell hybrids. Since the human retinoblastoma susceptibility gene (RB1) along with the gene for esterase D is on chromosome 13q14, these data indicate this linkage group is conserved in man and mouse.
Mouse melanoma growth stimulatory activity gene (Mgsa) is polymorphic and syntenic with the W, patch, rumpwhite, and recessive spotting loci on chromosome 5.
Melanoma growth stimulatory activity (Mgsa) is a polypeptide growth factor originally detected in culture medium of the human malignant melanoma cell line Hs294T and may have an autocrine role in neoplastic growth. Mgsa is a member of the small inducible gene (SIG) family and shares homology with beta-thromboglobulin and platelet factor 4. Mgsa was localized to chromosome 5 using a cDNA probe for mouse Mgsa and somatic cell hybrids and is thus syntenic with Kit (W), Ph, Rw, and rs loci. The results eliminate Mgsa as the product of the Steel locus on chromosome 10, but raise the possibility that Mgsa might be synonymous with a chromosome 5 locus affecting skin pigmentation.
Sigje, a member of the small inducible gene family that includes platelet factor 4 and melanoma growth stimulatory activity, is on mouse chromosome 11.
Stiles and coworkers originally identified a gene they termed JE that is transcriptionally activated in mouse fibroblasts early after treatment with platelet derived growth factor or serum. This gene, now named Sigje, can encode a 148-amino acid secreted, basic polypeptide that belongs to the small inducible gene (SIG) family whose members include, for example, platelet factor 4, melanoma growth stimulatory activity (Mgsa), and interferon inducible protein 10. SIG family members share a conserved array of cysteine and proline residues and a similar predicted secondary structure, and may have evolved from a common ancestral gene. Several members of the SIG family have been assigned to the proximal long arm of human chromosome 4, to a region that has genetic homoeology with a portion of mouse Chromosome 5. We report here that mouse Sigje, in contrast to Mgsa, is on Chromosome 11. Sigje restriction fragment length polymorphisms in Mus spretus DNA were identified with the enzymes TaqI, MspI, BclI, and XbaI, and will be useful in mapping by meiotic recombination.
Chromosome assignment of mouse insulin, colony stimulating factor 1, and low-density lipoprotein receptors.
Receptors for insulin, low-density lipoprotein, and colony stimulating factor 1 are associated with diabetes, atherosclerosis, and cancer in man. Complementary DNA clones for Insr, Ldlr, and Csfmr were used to chromosomally assign the three genes in mouse. In contrast to their close linkage on the short arm of human Chromosome 19, Insr and Ldlr are asyntenic, residing on mouse Chromosomes 8 and 9, respectively. The genes for CSF1R, CSF1, CSF2, IL-3, and IL-5 form a cluster on the long arm of human Chromosome 5. In mouse, Csfm, Csfgm, and IL-3 are syntenic on Chromosome 11. The Csfmr gene was assigned to mouse Chromosome 18 and is thus unlinked to other members of this gene cluster. These gene assignments provide additional topographical information on conservation of linkage groups in man and mouse and provide a genetic framework for evaluating the possible roles for the three receptor genes in genetic diseases in mouse.
Chromosomal assignments of genes for tissue plasminogen activator and urokinase in mouse.
The genes encoding the two plasminogen activators, tissue plasminogen activator and urokinase, were mapped to mouse chromosomes using probes derived from the respective mouse cDNAs. DNA from mouse-Chinese hamster and mouse-rat somatic cell hybrids was digested with BamHI and EcoRI, respectively, and analyzed by Southern blot hybridization for the segregation of the two genes. Tissue plasminogen activator and urokinase cosegregated with mouse chromosomes 8 and 14, respectively. The plasminogen activator genes thus fall into two syntenic groups that are conserved in human and mouse.
Unstable and stable CAD gene amplification: importance of flanking sequences and nuclear environment in gene amplification.
We analyzed the amplification of the CAD gene in independently isolated N-(phosphonacetyl)-L-aspartate-resistant clones derived from single parental clones in two mouse cell lines. We report for the first time that the CAD gene is amplified unstably in mouse cells, that the degree of instability varies greatly between clones, and that minute chromosomes and highly unstable chromosomelike structures contain the amplified sequences. These data are most consistent with the idea that the amplified unit in each clone consists of different flanking DNA and that such differences engender amplified sequences with unequal stability. We also introduced the mouse chromosome containing the CAD gene into hamster cells by microcell-mediated chromosome transfer to determine whether the propensity for unstable extrachromosomal amplification of the mouse CAD gene would prevail in the hamster cell nuclear environment. We report that the mouse CAD gene was amplified stably in expanded chromosomal regions in each of seven hybrids that were analyzed. This observation is consistent with the idea that the nuclear environment influences whether mutants containing intra- or extrachromosomally amplified sequences will be isolated.
Isolation of microcell hybrid clones containing retroviral vector insertions into specific human chromosomes.
We sought an efficient means to introduce specific human chromosomes into stable interspecific hybrid cells for applications in gene mapping and studies of gene regulation. A defective amphotropic retrovirus was used to insert the gene conferring G418 resistance (neo), a dominant selectable marker, into the chromosomes of diploid human fibroblasts, and the marked chromosomes were transferred to mouse recipient cells by microcell fusion. We recovered five microcell hybrid clones containing one or two intact human chromosomes which were identified by karyotype and marker analysis. Integration of the neo gene into a specific human chromosome in four hybrid clones was confirmed by segregation analysis or by in situ hybridization. We recovered four different human chromosomes into which the G418 resistance gene had integrated: human chromosomes 11, 14, 20, and 21. The high efficiency of retroviral vector transformation makes it possible to insert selectable markers into any mammalian chromosomes of interest.