Gerard Bittner

Tumor necrosis factor, but not other hematopoietic growth factors, prolongs the survival of hairy cell leukemia cells.

In order to determine the growth factor requirements of hairy cell leukemia (HCL) cells, we studied the in vitro effects of tumor necrosis factor (TNF), interleukin (IL) 1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, B-cell growth factor (BCGF), GM-CSF, PHA-stimulated lymphocyte-conditioned media (CM), and 5637 bladder carcinoma CM on HCL cells obtained from spleens of patients with HCL. Mononuclear cells from a normal donor, obtained at post-traumatic splenectomy, served as a control. TNF prolonged the survival of HCL cells obtained from five different HCL patients when compared to cells cultured in control media alone, although cell proliferation could be demonstrated in only two of the five. HCL cells stained negative for the Epstein-Barr nuclear antigen (EBNA) both before and after 4 weeks in culture. BCGF, 5637 CM, and PHA-stimulated lymphocyte CM also prolonged the survival of HC25 and HC56 cells, although not to the same degree as TNF. Cells cultured in BCGF, however, stained positive for EBNA. None of the other recombinantly produced or purified cytokines prolonged the survival of the leukemic cells. With the exception of IL-2, none of the growth factors studied prolonged the survival of purified normal spleen (NS) cells over a 4-week period of time when compared to NS cells incubated in media alone. TNF prolonged the survival of HC25 cells in a dose-dependent manner, and a highly purified antibody to TNF abrogated the effects of TNF. HC25 cells incubated in the presence of control media alone did not constitutively produce TNF mRNA; however, incubation of the cells in the presence of TNF for 48 h induced the cells to express TNF message. We conclude that TNF is important in prolonging the survival of HCL cells, and thus may be important in the pathogenesis of this disease.

Establishment and characterization of a SV40 T-antigen immortalized human bronchial epithelial cell line

Dear Editor: Events surrounding the transformation of normal human epithelial cells to the frankly malignant phenotype are being studied by numerous investigators. These studies are exemplified by reports describing the genetic events associated with the neoplastic development of colorectal carcinoma (3). The study of the transformation of normal human colonic epithelium to carcinoma has been facilitated by the availability of preneoplastic tissue in the form of adenomatous disease, which ranges from the hyperplastic state to premalignant. These tissues are readily available because of their ease of identification through colonoscopy, and the low morbidity related to that procedure. Studies of colonic tissue ranging from early adenomas, intermediate adenomas, late adenoma, to frank carcinoma have identified both activation of oncogenes and mutational inactivation of tumor suppressor genes, with the latter predominating (3). Unlike colorectal carcinoma, preneoplastic tissue is not readily available from human lung tissue. Although squamous metaplasia is thought to precede the development of lung cancer, this is a histological diagnosis, and not readily identifiable through a bronchoscope. Furthermore, bronchoscopy is associated with significant morbidity, and not done on a routine basis as a screening procedure. Thus, an in vitro model is necessary to identify the genetic changes associated with multistep bronchogenic carcinogenesis. We report the successful immortalization of normal human bronchial epithelial cells to an immortalized bronchial epithelial cell line by strontium chloride transfection of a plasmid carrying the replication origin defective large T antigen SV40 genome. Our objective was to establish an immortalized HBE cell line so as to have a culture system which would allow extended replication of HBE ceils, and thus facilitate the study of human lung tumor cell biology. A tracheobronchial specimen was obtained from a 20-year old female at the time of autopsy, and cultured as previously described (2,7). Briefly, the epithelial layer from human tracheobronchial specimens was separated from the underlying tissue with forceps, minced into approximately 1 mm 2 explants and plated (approximately 30 explants/100 mm dish) onto type I rat tail collagen-gelcoated dishes in approximately 1 ml of media. Ammonia-reconstituted collagen gels were pre-equilibrated in supplemented Hams F12 medium for at least 48 hours prior to use. The culture media consisted of Hams F12, supplemented with 2% FBS, insulin, transferrin, EGF, dextrose, hydrocortisone, and a mixture of nonessential amino acids. This supplemented medium, referred to as F 12 + 2 FBS, also contained Gentamycin and fungizone. Cultures were grown in humidified incubators at 37 ° C in an atmosphere of 5% CO2 and received media changes 2 to 3 times per week. Passage of explants was performed using repeated 4 minute exchanges of HEDH (Ca ++and Mg++-free HBSS with 0.02% EDTA, 8% dialyzed fetal bovine serum and 25 mM HEPES) at 37 o

Karyotypic changes associated with spontaneous acquisition and loss of tumorigenicity in a human transformed bronchial epithelial cell line: evidence for in vivo selection of transformed clones.

SummaryIn this study, we describe the karyotypic changes associated with the spontaneous acquisition of tumorigenicity in an immortalized tumor bronchial cell line. Neoplastic transformation of the NL20 human bronchial epithelial cell line occurred after 3 yr in culture, and was associated with loss of chromosome 18 together with acquisition of multiple copies of 9q21.2→34. The nontumorigenic NL20 cell line had been established by transfection of human bronchial epithelial cells with the SV40 T antigen, and had retained a relatively stable karyotype after the first 32 passages in vitro. However, when cells from p184 were inoculated into nude mice, a transplantable tumor was obtained that was derived from a minor clone present in this otherwise stable line. Subsequent passaging of the NL20 cells in vitro did not yield further tumors, and the minor clone from which the tumorigenic NL20T cell line derived was no longer evident in NL20 cells by Passage 205. Furthermore, the original tumorigenic NL20T cells lost the neoplastic phenotype after 25 passages in vitro and reverted to the nontumorigenic karyotype observed at p189. In contrast to the loss of the tumorigenic phenotype and karyotype, which occurred with in vitro passaging of the original tumor, when the NL20T cells were passaged in other nude mice, they continued to give rise to tumors with sevenfold amplifications of 9q sequences and loss of chromosome 18, and cells from the secondary tumors (NL20T-A cells) have maintained a stable karyotype and remain tumorigenic even after 64 passages in vitro. A mixture of 10% tumorigenic NL20T-A and 90% nontumorigenic NL20 cells formed tumors in athymic nude mice when cultured in vitro on fibronectin, but not on plastic; cytogenetic analysis demonstrated that the tumors and cell cultures were composed of tumorigenic NL20T-A cells, whereas cytogenetic analysis of cells cultured on plastic were identical to the nontumorigenic NL20 cells. These data support the hypothesis that neoplastic transformation in our original cell line arose from in vivo selection of a small mutant clone, which had arisen in culture and was subsequently selected in vivo but was lost with in vitro culture.

The Development of Techniques That Permit the Selection and Growth of Malignant Cells from Human Colonic Carcinomas

Publisher Summary This chapter discusses the development of techniques that permit the selection and growth of malignant cells from human colonic carcinomas. The technique which, was developed involved a human colon cancer cell line bank utilizing culture methodologies that permit successful establishment of cell lines from the majority of cancer specimens cultured. Cell lines have been established from neoplastic specimens which include the full spectrum of the clinical disease including an adenoma, localized and aggressive primary cancers, and metastases. These cell lines display biologic and pharmacologic diversity, making the VACO cell line bank an attractive disease-specific model for use in drug discovery programs as well as studies to understand the biologic progression of colon cancer. It was recognized that cell lines undergo selective pressures during in vitro cultivation and as a result may not retain all phenotypes present in the individual clinical tissue of origin. On the other hand, the preservation of such diverse characteristics in these established lines suggests that they exhibit at the least a portion of the diversity present in the patients, and they represent a valuable and practical model system for the laboratory investigation of colon cancer.