RESUMO
Double minutes (dm) have been isolated from human tumor cells by zonal centrifugation and by differential pelleting of chromosome suspsension. These methods allowed collection of dm in sufficient quantity and purity for visualization with electron microscopy. Ultrastructurally, the chromatin fibers in dm resemble thrance fragments was found. When the two isolation protocols were compared, differential pelleting was shown to increase purity twofold to 85% dm by mass. The differential pelleting procedure enables easy collection of dm in sufficient quantity and purity for chemical analysis.
Assuntos
Ciclo Celular , Núcleo Celular/ultraestrutura , Cromossomos/ultraestrutura , Metáfase , Carcinoma , Fracionamento Celular , Linhagem Celular , Centrifugação Zonal , Cromatina/ultraestrutura , HumanosRESUMO
Avian erythroblastosis virus has transduced two cellular genes, c-erb-A and c-erb-B. Using fractionated chicken chromosomes, we found that the two genes are located on different chromosomes in the chicken genome: c-erb-A is on a microchromosome, and c-erb-B is on a large chromosome. The locations of two other cellular oncogenes (c-fps and c-myb) were also determined: c-fps is on a microchromosome, and c-myb is on chromosome of an intermediate size. Our results suggest that avian erythroblastosis virus had transduced the two cellular genes independently, conforming to previous indications that cellular oncogenes are dispersed among multiple chromosomes in every species that has been examined.
Assuntos
Alpharetrovirus/genética , Vírus da Leucose Aviária/genética , Genes Virais , Oncogenes , Transdução Genética , Animais , Fracionamento Celular , Galinhas , Mapeamento Cromossômico , Cromossomos/ultraestrutura , Hibridização de Ácido NucleicoRESUMO
The in vivo tumorigenicity of malignant mouse-nonmalignant human somatic cell hybrids was correlated with the in vitro characteristics. The renal adenocarcinoma mouse cell line RAG and the normal, diploid human cell line Wl-38 were used as the fusion parents. Five independent RAG Wl 38 hybrid clones were tested for concanavalin A (Con A) agglutination patterns, in vitro invasiveness, and tumor formation in immunosuppressed mice. Tests on the parental lines showed that RAG cells agglutinated at much lower levels of Con A than the Wl-38 cells. RAG cells overgrew Wl-38 cells in the in vitro invasiveness assay. RAG cells readily formed tumors in untreated adult or young immunosuppressed mice, whereas the Wl-38 cells did not. The five hybrid clones were all similar, since they had Con A agglutination levels intermediate to those of both parents, though patterns were more "tumor-like", overgrew the Wl-38 cells in the invasiveness assay, and formed tumors in immunosuppressed mice.
Assuntos
Células Híbridas , Neoplasias Experimentais/etiologia , Adenocarcinoma/etiologia , Aglutinação , Animais , Divisão Celular , Cromossomos/ultraestrutura , Concanavalina A/farmacologia , Humanos , Células Híbridas/enzimologia , Terapia de Imunossupressão , Neoplasias Renais/etiologia , Masculino , CamundongosRESUMO
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.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 10 , Glioblastoma/genética , Glioblastoma/patologia , Polimorfismo de Fragmento de Restrição , Animais , Linhagem Celular , Sobrevivência Celular , Mapeamento Cromossômico , Humanos , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Nus , Transplante Heterólogo , Células Tumorais CultivadasAssuntos
Divisão Celular/efeitos dos fármacos , Cílios/efeitos dos fármacos , Colchicina/farmacologia , Animais , Núcleo Celular/efeitos dos fármacos , Cromossomos/efeitos dos fármacos , Cricetinae , Fibroblastos/metabolismo , Complexo de Golgi/efeitos dos fármacos , Masculino , Microscopia EletrônicaRESUMO
As the molecular details of cancer have begun to unfold, it has become apparent that the cellular genetic apparatus is defective in the malignant cell. Accordingly, we have attempted to review the prominent aspects of genetics research as it impinges on the problem of cancer. Although the field is immense, we have tried to cover four major areas: human genetics, molecular genetics, somatic cell genetics, and developmental genetics. Oncogenes are considered in detail in all of these areas, and a new map (42 entries) of oncogene positions on human chromosomes is presented.
Assuntos
Neoplasias/genética , Bandeamento Cromossômico , Cromossomos Humanos , Feminino , Amplificação de Genes , Humanos , Masculino , Biologia Molecular , Oncogenes , Translocação GenéticaRESUMO
The right of incarcerated prison and jail inmates to health care is protected by the 8th and the 14th amendments of the Constitution, respectively. Does the right to health care include access to clinical trials? At the time of this writing, clinical trials have become part of the fabric of HIV/AIDS care, allowing patients to participate in studies of new and often lifesaving treatments. Participation in trials can also be dangerous, as illustrated by the recent death of a subject in a gene therapy trial. This danger is compounded by ethical dilemmas that can arise from the large amount of financial support for clinical trials (greater than 75%) that is derived from for-profit corporations. Indeed, clinical trials are the subject of grave concern on the part of the United States Government, which has recently taken steps to shore up human subject safeguards. Following a conference on the conduct of clinical trials in correctional settings, the Office for Human Research Protections suspended prison research conducted by 4 prestigious academic institutions.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Ensaios Clínicos como Assunto , Infecções por HIV/tratamento farmacológico , Guias de Prática Clínica como Assunto/normas , Prisioneiros , Ética , Acessibilidade aos Serviços de Saúde/legislação & jurisprudência , Acessibilidade aos Serviços de Saúde/normas , Experimentação Humana , Humanos , Estados UnidosAssuntos
Cromossomos , Fibroblastos , Animais , Aberrações Cromossômicas , Transtornos Cromossômicos , Cricetinae , Citogenética , Técnicas In Vitro , CariotipagemRESUMO
Isolated Chinese hamster chromosomes dissociate into a series of specific chromatin subunits approximately the size of stainable chromosome bands upon reduction of the divalent ion concentration during or after isolation. At high pH the chromatin in some bands is differentially removable during chromosome isolation, leaving a banded chromosome with a pattern typical of most G-band procedures. This provides an alternate molecular mechanism to explain the production of banded chromosomes by a variety of staining procedures. These results also suggest an approach to chromatin fractionation, using metaphase chromosomes as a starting material.
Assuntos
Cromatina/ultraestrutura , Cromossomos/ultraestrutura , Animais , Cátions Bivalentes , Linhagem Celular , Bandeamento Cromossômico , Cricetinae , Cricetulus , Concentração de Íons de Hidrogênio , Metáfase , Microscopia Eletrônica , Modelos Biológicos , Coloração e RotulagemRESUMO
A new genetic mechanism is proposed to explain the evident order seen in embryonic development. This theory postulates control DNA, a set of genetic elements activated in a specific sequence, one at a time. With each cell division, control of gene expression passes to the next control unit in the series. The complete series of control units would constitute the encoded (and inherited) development program of an organism.
Assuntos
Desenvolvimento Embrionário e Fetal , Genes , Código Genético , Animais , Caenorhabditis , Divisão Celular , Cordados não Vertebrados , DNA/genética , Replicação do DNA , Drosophila , Indução Embrionária , Regulação da Expressão Gênica , Modelos GenéticosRESUMO
Chinese hamster fibroblasts were synchronized and given 5-bromodeoxyuridine for DNA synthesis except during one hour of the S phase when thymidine was present in the medium. In the next mitosis, chromosomes stained with 33258 Hoechst were banded in appearance when photographed by fluorescence microscopy. The bright regions corresponded to the chromosome segments replicated during the thymidine exposure in the S phase. The segments replicated together during any one hour produced three distinct patterns which were characteristic of early, middle, and late S phase. Most of the fluorescent regions corresponded in size and position with G-bands of these chromosomes. There was no correlation between the staining behavior of a band in G-band procedure and its time-or-replication, i.e., both light and dark G-bands were replicated during early, middle, and late S phase. However, it appears that all of the DNA within a single band is replicated together within one third of the S phase.
Assuntos
Cromossomos/metabolismo , Replicação do DNA , Animais , Benzimidazóis , Bromodesoxiuridina/metabolismo , Cricetinae , Demecolcina/farmacologia , Corantes Fluorescentes , Mitose , Cromossomos Sexuais/metabolismo , Coloração e Rotulagem , Timidina/metabolismo , Fatores de TempoRESUMO
Microcells, cytoplasmic fragments that contain micronuclei composed of one or a few chromosomes, can be generated directly from mitotic cells. Cytochalasin B, which causes nuclear extrusion in interphase cells, has a similar effect on the chromosomes of colcemid-blocked mitotic cells. The forces generated during centrifugation in a Percoll gradient are sufficient to separate the extruded microcells from the parent cell. The chromosomes contained in an extruded microcell form micronuclei during the process, and in all respects are comparable to microcells generated from micronucleated cells except that they are uniformly in the G1 phase of the cell replication cycle. The procedure is probably applicable to all mammalian cells that grow in culture and can be employed to make microcells for the transfer of both intact and fragmented chromosomes.
Assuntos
Fracionamento Celular , Cromossomos , Mitose , Animais , Núcleo Celular , Células Cultivadas , Centrifugação com Gradiente de Concentração , Cricetinae , Citocalasina B/farmacologia , Fibroblastos/efeitos dos fármacos , Humanos , Povidona , Dióxido de SilícioRESUMO
DNA-mediated gene transfer has been used successfully in many experiments to identify and isolate transforming sequences from human tumors and tumor cell lines. This work was done to compare the efficiency of that technique to microcell-mediated chromosome transfer in the transformation of NIH 3T3 mouse fibroblast cells. Our hope was that oncogenes introduced into a cell in chromosomal form would also be effective in the transformation of NIH 3T3 cells. The study revealed, however, that microcells from cell lines that contain transforming sequences identified by DNA-mediated gene transfer were unable to transform NIH 3T3 cells, although other human genes were expressed in the microcell hybrids. Several possible mechanisms are given and discussed. This research may provide important insight into the possible control of oncogenes in intact human tumor cells.
Assuntos
Oncogenes , Transfecção , Animais , Transformação Celular Neoplásica , Células Cultivadas , Sondas de DNA , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Humanos , Células Híbridas/citologia , Cariotipagem , Camundongos , Células Tumorais Cultivadas/citologiaRESUMO
DNA synthesis in a broken cellular preparation of Chinese hamster cells was enhanced approximately 10-fold by a brief trypsin treatment. Alphachymotrypsin also enhanced the synthesis, whereas Pronase did not. The trypsin appears to be acting on a component of the nucleus. Evidence suggests that the trypsin is not removing protein from the DNA, but may be activating the system some other way.
Assuntos
DNA/biossíntese , Tripsina/farmacologia , Animais , Linhagem Celular , Sistema Livre de Células , Quimotripsina/farmacologia , Cricetinae , Fibroblastos , Cinética , Pronase/farmacologia , Estimulação QuímicaRESUMO
We have developed a general technique for making micronucleated cells to use in microcell-mediated chromosome transfer. Growing cells are blocked in mitosis with colcemid, placed in a hypotonic solution for 10 min, and returned to culture medium for 24 h. This treatment promotes the formation of micronuclei within lymphoblast or fibroblast cells. The microcells are generated by cytochalasin B treatment on a Percoll density gradient centrifuged at 43,500g. The resulting mixture of microcells, whole cells, and karyoplasts is filtered through 3-micron pores to obtain a pure microcell preparation. The microcells are fused to recipient whole cells using phytohemagglutinin-P and polyethylene glycol. Advantages of this technique are: donor cells need not be attached to a substrate; and cell lines which form micronuclei in low frequency can still be used efficiently as microcell donors.