Helen Thornton

Heterotransplantation of human glioblastoma multiforme and meningioma to nude mice.

Summary A human glioblastoma multi-forme (Br7) and a meningioma have been successfully xenografted sc in nude mice. Both were successfully passed to a second generation. Complete retention of the unique histologic features was noted in each tumor xenograft. Actively growing cultures from the glioblastoma xenograft have been established; they are composed of the cell types characteristic of glioblastoma. Immature and mature C-type particles, presumably representing a xenotropic mouse virus, are present in both grafts. The growth of these two tumors in nude mice may provide a useful model for etiologic, immunologic, and chemotherapeutic studies. An attempt to produce glioblastoma in nude mice by the injection of cells cultured from human glioblastoma T98 was unsuccessful.

Oncogenes: their presence and significance in squamous cell cancer of the head and neck.

DNA extracted from squamous cell carcinomas of the larynx and tongue has been shown to contain cellular transforming genes characterized by their ability to transform mouse fibroblasts into malignant foci of cells which, when subsequently cloned and grown to volume, have been found to contain human DNA sequences. This DNA has been serially passaged through subsequent populations of NIH/3T3 mouse fibroblasts. Higher malignant transformation efficiencies have been observed and reported with serial passage. Of greater significance is the repeated identification of oncogenes of identical characteristics on electrophoretic radioisotope analysis.

Oncogenes: preliminary studies in head and neck cancer.

DNA has been extracted from squamous cell carcinomas of the larynx, base of tongue, and nasopharynx. These tumors were excised from patients at the St. Louis University Medical Center and processed at the Institute for Molecular Virology of the St. Louis University Medical Center. NIH/3T3 cells were transfected with the DNAs from these cancers. Malignant, transformed foci of NIH/3T3 cells have been observed. These foci have been cloned and grown in quantity. The cloned foci have been injected into nude mice with the production of highly malignant sarcomas. DNA extracted from these sarcomas has shown homology with human DNA on hybridization analyses of both nasopharynx and tongue cancer. Further hybridization studies are being conducted on the larynx cancer‐induced sarcomas and on the DNAs taken from the original transformed foci of NIH/3T3 cells transfected with squamous cell cancers of the larynx, nasopharynx, and tongue base. Our preliminary results indicating the presence of human sequences in the mouse sarcomas support the hypothesis that human cellular transforming gene(s) may be present in the DNA isolated from the head and neck cancers. Additional studies will include repetitive retransfection of NIH/3T3 cells, molecular cloning of putative oncogenes, and DNA sequence analysis of the cloned oncogenes. It is hoped that identification of putative oncogene sequences will result in the identification of the proteins coded for by the specific nucleotide sequences responsible for malignant cellular transformation by DNA extracted from head and neck tumors.

Studies on virus induction by 5-bromodeoxyuridine in nonproducer murine sarcoma virus-transformed 3T3 cells

Abstract Treatment with 5-bromodeoxyuridine (BrdU) of nonproducer BALB 3T3 cells (KA31 cells) transformed by the Kirsten strain of murine sarcoma virus (Ki-MSV) induced the synthesis of DNA polymerase-containing particles which band in sucrose gradients at a density of 1.16 and utilize poly(I) (dC) 12–18 , a template-primer specific for RNA-directed DNA polymerase. In order to establish optimal conditions for virus induction, the induction process was studied by analysis of the culture fluid for polymerase activity and the induced cells for viral proteins by immunofluorescence and virus particles by electron microscopy. By varying the concentration and time of exposure to BrdU, it was found that the maximal level of polymerase activity was induced when cells were treated with 20 μg of BrdU for a 24-hr period, 24 hr after seeding. Under these conditions about 15% of the cells were induced to synthesize viral antigens detected with anti-Moloney MSV rat serum. The level of polymerase activity in the culture fluid and viral antigen in the cells became maximal at 3 days after BrdU treatment and decreased rapidly. Electron microscopy showed a large increase in the proportion of cells containing incomplete virus particles and in the number of particles per cell at 2 and 3 days after BrdU treatment. At 3 days after treatment, 82% of the cells contained incomplete virus particles within dilated vesicles of the endoplasmic reticulum as compared to 18% for untreated cultures. A high level of virus production could be maintained for at least 15 days by continuous maintenance of cells on 4 μg/ml of BrdU. It is not clear whether the mechanism involves continuous reinduction of virus or overcoming the normal inhibition of virus multiplication in these cells. Under these conditions, BrdU reversed the rounded, transformed appearance of KA31 cells to a flat fibroblastic morphology.

Oncogenes in laryngeal cancer: serial passage of transformed cellular DNA.

DNA originally extracted from squamous cell cancer of the larynx has been serially passaged through transformed populations of NIH/3T3 mouse fibroblasts. The transformed foci were then harvested, cloned to volume, and incubated with a fresh population of NIH/3T3 cells in a second passage. Transforming efficiencies were enhanced by serial passage. In addition, Southern Blot analysis of the transformed foci revealed hybridization between transformant DNA and human probe DNA from the Alu family of conserved human DNA sequences. In the first passage this hybridization took the form of diffuse homology throughout the entire molecular weight distribution. The second-passage DNA showed “narrow bands” indicating the possibility that an oncogene has been identified in laryngeal cancer and that serial passage has eliminated contaminating human sequences. Repetitive transfection in third- and fourth-passage studies is now being completed.