RESUMEN
alpha-Acetoxy-N-nitrosodimethylamine, an activated derivative of the carcinogen N-nitrosodimethylamine, methylated in vitro a plasmid containing the human c-Ha-ras-1 proto-oncogene, resulting in the generation of a transforming oncogene, assayed by transfection into NIH 3T3 cells. The resulting transformed cells were tumorigenic and metastatic in immune-deprived mice. Further transfection using tumor DNA led to the formation of three secondary NIH 3T3 transformants. DNA from these secondary transformants contained human ras gene sequences. Two of the three secondary transformants contained G----A mutations at guanine 35 in codon 12, and the third secondary transformant retained the wild-type sequence at codons 12, and 61. For the latter, the activating mutation was not determined. These results demonstrate that a simple methylating agent can activate a normal human ras proto-oncogene to a transforming oncogene.
Asunto(s)
Dimetilnitrosamina/análogos & derivados , Regulación de la Expresión Génica , Genes ras/efectos de los fármacos , Animales , Southern Blotting , Pruebas de Carcinogenicidad , Línea Celular , Transformación Celular Neoplásica , ADN/metabolismo , Análisis Mutacional de ADN , ADN Polimerasa Dirigida por ADN , Dimetilnitrosamina/farmacología , Amplificación de Genes , Humanos , Masculino , Metilación , Ratones , Ratones Endogámicos CBA , Sondas de Oligonucleótidos/síntesis química , Sondas de Oligonucleótidos/genética , Plásmidos , Proto-Oncogenes Mas , Polimerasa Taq , TransfecciónRESUMEN
The human bladder cancer cell line MGH-U1 (also designated T-24 or EJ) contains an activated c-Ha-ras oncogene, which is amplified as compared to normal human fibroblasts. We have generated sublines from the MGH-U1 cell line: the MGH-U1/OCI subline was generated by dissociating spheroids formed from MGH-U1 cells; the U1-m/F1 and OCI-m/F1 were generated by in vivo passage of experimental lung metastases formed after i.v. injection of MGH-U1 and MGH-U1/OCI lines into immune-deprived mice; the U1/t subline was generated by in vivo passage of i.m. tumors formed from MGH-U1 cells. All sublines formed tumors in immune-deprived mice from smaller i.m. inocula than the parent line, and the U1-m/F1 subline generated more spontaneous metastases in lungs. Lung colony forming efficiency after i.v. injections of cells into similar mice was also greater for the sublines than for the parent MGH-U1 cells. The U1-m/F1 and OCI-m/F1 were the most tumorigenic lines. Early passages of the MGH-U1/OCI subline showed the presence of double minute chromosomes, and amplification and increased expression of the c-Ha-ras oncogene as compared to the parental cell line. These changes were not present in later cultures of MGH-U1/OCI cells, and no consistent difference in the levels of gene amplification or expression between the parent line and the sublines was found. Thus the content and expression of the activated c-Ha-ras oncogene does not correlate with malignant properties of the sublines.
Asunto(s)
Proto-Oncogenes , Neoplasias de la Vejiga Urinaria/patología , Animales , Transformación Celular Neoplásica , Amplificación de Genes , Humanos , Cariotipificación , Masculino , Ratones , Ratones Endogámicos CBA , Metástasis de la Neoplasia , Trasplante de Neoplasias , Trasplante Heterólogo , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/inmunologíaRESUMEN
Mice that are immune-suppressed by thymectomy and by sequential treatment with 1-beta-D-arabinofuranosylcytosine and whole body irradiation may be used as hosts for generation of human tumor xenografts. We have studied the effect of various additional methods of immune suppression on the formation of tumors after i.m. injection and on the formation of lung colonies after i.v. injection with the human MGH-U1 bladder cancer cell line. Success of transplantation was improved by treatment of immune-suppressed animals with either heterologous antilymphocyte serum or a monoclonal anti-Thy-1.2 antibody. Success of lung colony formation was also improved by antilymphocyte serum but not by monoclonal anti-Thy-1.2 antibody. Admixture of heavily irradiated cells (10(6)) to the viable inoculum of tumor cells in addition to antilymphocyte serum treatment improved the success of i.m. transplantation but not that of lung colony formation. Treatment with corticosteroids or treatment with carrageenan to suppress macrophage activity added toxicity and did not improve the success of xenografting. Immune suppression decreased the natural killer cell activity of normal mice and treatment with antiinterferon to further suppress natural killer cells may also enhance xenograft formation. Administration of cyclosporin A to normal mice allowed the growth of a single xenograft but was not a useful method for immunosuppression. The success of xenografting into immune-deprived mice was superior to that for two strains of nude mice maintained in our laboratory, and i.v. injection of tumor cells did not lead to lung colonies in these nude mice. Immune-deprived mice are a useful alternative to nude mice for the study of xenografts derived from human tumor cell lines and may allow the study of experimental lung metastases.
Asunto(s)
Terapia de Inmunosupresión/métodos , Neoplasias Pulmonares/secundario , Trasplante de Neoplasias , Neoplasias Experimentales/patología , Animales , Suero Antilinfocítico/farmacología , Carragenina/farmacología , Ciclofosfamida/farmacología , Ciclosporinas/farmacología , Citarabina/farmacología , Estradiol/farmacología , Femenino , Células Asesinas Naturales/inmunología , Masculino , Metilprednisolona/farmacología , Ratones , Ratones Endogámicos CBA , Timectomía , Trasplante Heterólogo , Irradiación Corporal TotalRESUMEN
Seventy-four biopsies of human bladder carcinoma were assessed by implantation as xenografts in immune-deprived mice and/or by culture of cell suspensions in agar or methylcellulose. The quality of the cell suspensions was assessed immediately after plating in vitro. The results were compared with the pathological stage and grade of the biopsies and with the clinical course of the disease in patients from whom the biopsies were obtained. We found that (a) progressively growing xenografts were generated from 20 of 53 biopsies (38%). These xenografts grew with mean volume doubling times in the range of 1 to 3 weeks; all of them examined histologically were consistent with transitional cell carcinoma. (b) Colony formation occurred from 21 of 49 cell suspensions (43%), and plating efficiency was in the range of 0.0004 to 1.7%. The majority of cell suspensions were found to have residual small clusters of cells. Colony formation sometimes originated from these clusters, an effect that would be expected to introduce artifacts when the in vivo cloning assay is used for chemosensitivity testing. (c) There was no evident correlation between expression of clonal growth in vitro and success of xenografting, and no correlation between the results of either of these experimental procedures with stage, grade, or clinical course of the disease. Further improvements in tissue culture and xenograft technology will be required before these methods can be used as a guide to patient management.
Asunto(s)
Carcinoma de Células Transicionales/fisiopatología , Neoplasias de la Vejiga Urinaria/fisiopatología , Animales , División Celular , Células Cultivadas , Citarabina/toxicidad , Humanos , Masculino , Ratones , Ratones Endogámicos CBA , Trasplante de Neoplasias , Trasplante Heterólogo , Irradiación Corporal TotalRESUMEN
Human bladder cancer from cystoscopic biopsies and from established cell lines was transplanted into mice that were immune suppressed by thymectomy plus sequential treatment with 1-beta-D-arabinofuranosylcytosine and whole-body irradiation. Each of four established human bladder cancer cell lines generated transplantable tumors in these mice, and some of the mice developed pulmonary metastases. Eight of 33 cystoscopically obtained biopsies of transitional cell carcinoma and one from a metastatic site led to xenografts that grew progressively, and some of these have been transplanted and/or have generated cell lines in vitro. Xenografts grew after a lag period of 0 to 32 weeks and had doubling times of 9 to 30 days. All of those examined histologically were consistent with transitional cell carcinoma, but some of the xenografts became more or less well differentiated in first and subsequent passages. The immune-deprived mouse is an alternative host to the nude mouse for generation of human tumor xenografts and may be a useful model for study of biological properties and therapeutic response of human bladder cancer.