Induction of endogenous xenotropic retrovirus expression by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate

Summary The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined for its ability to induce endogenous retrovirus from a high-passage clone of Kirsten sarcoma virus-transformed Balb/c (K-Balb) mouse cells. TPA activated virus in a concentration-dependent manner (0.0016 to 4.0 μM). Exposure to 1mM actinomycin D inhibited virus induction, suggesting that cellular RNA synthesis is required de novo by this inducer. A broad-spectrum neutralizing antibody to murine type C virus, gp70, was shown to neutralize the infectivity of the induced virus. The activated virus had the host range of the xenotropic Balb virus:2, and after removal of the inducer, the activated state decayed rapidly. TPA stimulated DNA, RNA, and protein synthesis in K-Balb cells, indicating that the mechanism of inducation may be different from that of previously identified virus inducers. The effects observed using the well-defined K-Balb system offer an opportunity to study the modulation of retrovirus gene expression by TPA. This work was conducted while the authors were with the Biological Carcinogenesis Program, Frederick Cancer Research Facility, Frederick, MD 21701, and was supported under Contract NO1-CO-75380 with the National Cancer Institute, National Institutes of Health, Bethesda, MD 20205.     

Characteristics ofHerpesvirus saimiri-induced lymphoma cells in tissue culture

Summary Lymphoblastoid cells were cultured from twoHerpesvirus saimiri (HVS) inoculated white-lipped marmosets and from one HVS-inoculated owl monkey. Cells from all three animals grew clumped in suspension. The cells from both species were diploid in chromo-some number and showed no unusual chromosomal abnormalities. The marmoset cell line examined was chimaeric. The marmoset cells lacked HSV-associated antigens as determined by immunofluorescence, and no evidence for the presence of virus was found by either infectivity assays or electron microscopy. Cocultivation of these cells with Vero cells resulted in cytopathology and the recovery of complete, infectious virus. The owl monkey lymphoid cells were positive to a small degree for both viral antigens and infectivity. The cells were resistant to rechallenge with HVS. Cocultivation of these cells with Vero cells led to the development of cytopathology and an increased yield of virus. This work was supported in part by Contract NIH-NCI-E-71-2025 from the Special Virus Cancer Program, National Cancer Institute, National Institutes of Health, United States Public Health Service.     

Establishment of duck cell line derived from experimental tumor induced by 20-methylcholanthrene

Summary Experimental tumors developed in white Pekin ducks after intramuscular implantation of 20-methylcholanthrene. Cells derived from the primary tumor were adapted successfully to grow in vitro and have growth characteristics similar to that of established cell lines of mammalian origin. The cell density rises rapidly and the doubling time is approximately 19 hr. The duck cells have been cultured succesfully for at least 80 passages in vitro. The continuously cultured cells have the characteristic chromosome pattern of duck, and the DNA of the duck cell line hybridized with duck liver DNA. We believe we have established a continuous cell line of avian origin. Electron-microscopic examinations of the tumor cells and RNA-directed DNA polymerase of the cell-free supernate show no evidence of endogenous virus production. This study was supported by Public Health Service Research Grants CA 16479 and CA 20012 from the National Cancer Institute and RR 00890 from the National Institutes of Health.     

Development,characterization, and viral susceptibility of a feline (Felis catus) renal cell line (CRFK)

Summary Cell line CRFK, derived from kidney tissue of a normal domestic kitten, was initiated in 1964. With intermittent periods of storage in the frozen state, it has been grown in vitro during more than 200 passages, without apparent loss of susceptibility to selected viruses. Various herpesviruses and feline viruses belonging to differnet virus groups grow readily and with distinct, cytopathic features. The cells now grow as a smooth monolayer of epithelial-like cells; most have 37 chromosomes (2n−1) and are thus aneuploid for cat karyotype. Three distinct marker chromosomes are identified. The cell line, which is free of mycoplasmal contaimination, is useful in feline virus research and diagnostic medicine and has become of particular interest in cancer research. Supported in part by Contract E73-2001-NO1-CP-3-3237 within the Special Virus Cancer Program, National Cancer Institute, National Institutes of Health, Public Health Service and the Morris Animal Foundation and General Research Support funds of the New York State Veterinary College. CRFK cells from stock provided by C. G. Fabricant are available for distribution to investigators from the Cell Culture Laboratory, Naval Biomedical Research Laboratory.     

Intercellular communication and tissue growth

Summary Epithelial cells of normal rat (adult) liver and hamster embryo in tissue culture communicate through membrane junctions: the membrane regions of cell contact are highly ion-permeable. Cancerous counterparts of these cells, cells from Morris' and Reuber's liver tumors and from x-ray-transformed embryo cultures, do not communicate under the same experimental conditions. These cells also fail to communicate with contiguous normal cells. Cancerous fibroblastic cells from a variety of tissues, including cells transformed by virus, x-radiation and chemicals, communicate as well as their normal counterparts; this is so for long- and short-term cell cultures. Communication in some fibroblastic cells is sensitive to components of blood serum: normal and transformed hamster embryo fibroblasts, which communicate when cultured in medium containing fetal calf serum, appear to lose communication in medium containing calf serum; the converse holds for hamster (adult) fibroblasts and 3T3 cells.The preceding papers of this series appeared in the Journal of Cell Biology.Trainee of the National Institutes of Health, National Cancer Institute, Grant CA 05011.     

Aminocentesis cells: Culture,cryogenic storage,isozymes, and finite life in vitro

Summary Forty-six cell cultures established from amniocentesis fluids were preserved in liquid nitrogen and later recovered from the frozen state with little loss of viability as compared to prefreeze viability. Five to 10% glycerol was found to be optimal for preservation in liquid nitrogen, and as few as 5×10⁵ viable cells per frozen ampule could initiate cell growth. Storage in liquid nitrogen did not affect the genetic stability of glucose-6-phosphate dehydrogenase, lactate dehydrogenase, malic dehydrogenase, leucine aminopeptidase, acid phosphatase, or 6-phosphogluconic acid dehydrogenase isozymes of the amnion cultures. These studies were supported by Contract NIH-NIGMS-72-2070, Grant CA-04953-13 from the National Cancer Institute; General Research Support Grant FR-5582 from the National Institutes of Health; and Grant-in-Aid Contract M-43 from the State of New Jersey. Recipient of Research Career Award 5-K3,16, 749 from the National Institutes of Health.     

Quantitation of human mammary epithelial antigens in cells cultured from normal and cancerous breast tissues

Summary A sensitive radioimmunoassay technique was developed to quantitatite the level of human breast celltype specific antigens on cells from normal breast and from various established cell lines of breast and nonbreast origins. Polyacrylamide gel electrophoresis revealed four major proteinaceous components (150,000; 75,000; 60,000; and 48,000) in human milk fat globule membranes that were used to immunize rabbits in order to elicit antimammary epithelial cell antibody. Antisera obtained were rendered specific by abosorptions and were able to recognize three specific mammary epithelial components of the breast epithelial cell. Human mammary epithelial (HME) antigen expression was highest (1290 ng/10⁶ cells) in normal breast epithelial cells from primary cultures of normal breasts. Lower levels (range: 955 to 330 ng/10⁶ cells) were found in breast epithelial cells from cell lines established from cancerous breast tissue. Cells of nonbreast origins as well as fibroblasts from breast gave much lower values (less than 30 ng/10⁶ cells). On treatment, with trypsin, of two breast epithelial cell lines (MDA-MB-157 and MCF-7) 80 to 85% of their HME antigen expression was lost, suggesting that a majority of these breast antigens reside on the cell surface. This work was Supported by Grant PTD-99 from the American Cancer Society, Grant CA19455 and CA20286 from the National Cancer Institute, and Biomedical Research Support Grant RR05467 from the National Institutes of Health. Most cells used in the present study were produced with support from National Cancer Institute Contract Y01-CP8-0500, Biological Carcinogenesis Branch, Division of Cancer Cause and Prevention, under the auspices of the Office of Naval Research and the Regents of the University of California.     

Chromosomes of the murine leukemia virus indicator cell line XC

A cell line derived from the Rous Sarcoma Virus induced rat tumor XC (Svoboda), which was recently utilized as an indicator for the presence of murine leukemia virus growing in mouse cells, has been examined karyologically. The cells differ considerably from each other as well as from the normal rat karyotype (Rattus norvegicus, 2n=42). The modal chromosome number is 41. All cells bear one or more chromosome markers in common as well as non-rat-like chromosomes, but rat-like chromosomes still preserve the identity of species origin.Supported by Contract No. PH 43-63-13 between the University of California and the National Cancer Institute, National Institutes of Health (Special Virus Cancer Program).     

Species identity of insect cell lines

Summary Three mosquito cell cultures designated as Suitor's clone ofAedes aegypti, Culiseta inornata, andAedes vexans were shown to be moth by immunological, karyological, and isozyme analyses. The cells reacted with rabbit antimoth serum but not rabbit antimosquito serum. Chromosome analyses indicated Lepidopteran rather than Dipteran morphology, and three isozyme systems were confirmative. Any one of these assays would be sufficient to indicate that contamination had occurred and could be used as a periodic check for identity of cell cultures. Morphology and growth characteristics are also valid criteria to distinguish between these particular orders of insect cells. These studies were supported by Grant CA-04953-12 from the National Cancer Institute; General Research Support Grant FR-5582 from the National Institutes of Health; and Grant-in-Aid Contract M-43 from the State of New Jersey. Recipient of Research Career Award 5-K3-16,749 from the National Institutes of Health.     

Human leukemic cells: Cytochemical studies on nuclear proteins

The DNA:histone ratios have been determined by quantitative cytochemical analyses of individual cells in populations of human lymphocytic cells derived in continuous culture from the peripheral blood buffy coats of patients with acute leukemia or infectious mononucleosis. These populations of lymphocytic cells were quite similar with respect to the Feulgen-DNA and protein content per cell. The close association between DNA and histone was reflected in their similar patterns of distribution in fixed and stained cells; and further evidenced by similarities in the DNA: histone ratios characteristic of these different populations of lymphocytic cells. — Chemical acetylation and methylation of nuclear proteins of these cell populations exhibited some quantitative differences. The chemically acetylated histone content was less, and chemically methylated histone content was greater in cells derived from acute leukemia or infectious mononucleosis than in normal human lymphocytes. These quantitative differences in chemical acetylation and methylation may contribute to specific structural alterations in these histones which modify their functional capacity with respect to interactions with DNA. Such alterations may relate to differences in gene expression as reflected, for example, by the biological and biochemical differences among these human lymphocytic cells.These studies were supported in part by research grants C-6516 from the National Cancer Institute and FR-05526 from the Division of Research Facilities and Resources, National Institutes of Health.Holds Research Career Award K6-CA-22,150 from the National Cancer Institute, National Institutes of Health.     

The molecular biology computer research resource.

Described is a new National Institutes of Health supported Molecular Biology Computer Research Resource located at the Dana-Farber Cancer Institute in association with Harvard University, Boston, Massachusetts.     

Transformed phenotype conferred to NIH/3T3 cells by ectopic expression of heparin-binding growth factor 1/acidic fibroblast growth factor

Summary Heparin-binding growth factor 1 (HBGF-1), also known as acidic fibroblast growth factor, is a potent mitogen and angiogenic factor found in tissues such as brain, kidney and heart. The genomic and cDNA sequences indicate that HBGF-1 does not have a typical signal peptide sequence. HBGF-1 was shown to be localized to the extracellular matrix of cardiac myocytes, but the mechanism of secretion is not presently known. We have cloned the HBGF-1 cDNA which allowed us to directly test the biological activity, mechanism of secretion and transforming potential of the recombinant protein. A previous report showed that the truncated HBGF-1 confers partial transformed phenotype to the recipient fibroblasts. However, expression of full-length HBGF-1 has not been reported. The HBGF-1 coding sequence was cloned into the retroviral expression vector, SVX, and transfected into NIH/3T3 cells. Transfectants expressing full-length HBGF-1 protein at high levels form foci and grow to a higher cell density than the parental NIH/3T3 cells. Western blotting analysis showed that the recombinant HBGF-1 is a unique band of approximately 20 kDa and can be detected in the cell homogenate but not in the conditioned medium. NIH/3T3 cells were conferred anchorage independence when HBGF-1 was provided exogenously. We showed the transformed cells are capable of growing on soft agar even in the absence of exogenously-provided HBGF-1. Transfected cells expressing HBGF-1 also induced tumor formation when injected into nude mice. Thus, NIH/3T3 cells acquired a full spectrum of transformed phenotype when full length HBGF-1 was expressed at high levels. This work was supported by grants from the National Cancer Institute (RO1 CA45611), The March of Dimes Birth Defects Foundation (No. 6-549) and The Ohio Cancer Research Associates, Inc. I.-M.C. is a recipient of The Research Career Development Award (KO4 CA01369) from the National Institutes of Health.     

Persistent infection with bovine herpesvirus-1 (infectious bovine rhinotracheitis virus) in cultured hamster cells

Summary Bovine herpesvirus-1 infection in hamster embryo cells was found to be dependent upon input multiplicity; productive infection was achieved at input multiplicities greater than one, while persistent infection was established when input multiplicities were about 0.5. This persistence was characterized by a noncyclic, minimal degree of cytopathic effect with a low level of released virus. Maintenance of the persistently infected cultures did not require external supportive measures. Subcultivation of the persistently infected cultures led to virus replication followed by CPE and then cell regrowth. Within 3 to 4 weeks after subcultivation a persistent infection was re-established. The possible mechanism for the bovine herpesvirus persistence in hamster cells is discussed. This work was supported in part by Public Health Service Research Contract FDA 233-74-1035 and by Research Grant AI-08648 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.     

Species identity of insect cell lines

Summary Three mosquito cell cultures designated as Suitor's clone ofAedes aegypti, Culiseta inornata andAedes vexans were shown to be moth by immunological, karyological, and isozyme analyses. The cells reacted with rabbit antimoth serum but not rabbit antimosquito serum. Chromosome analyses indicated Lepidopteran rather than Dipteran morphology, and three isozyme systems were confirmative. Any one of these assays would be sufficient to indicate that contamination had occurred and could be used as a periodic check for identity of cell cultures. Morphology and growth characteristics are also valid criteria to distinguish between these particular orders of insect cells. These studies were supported by Grant CA-04953-12 from the National Cancer Institute; General Research Support Grant FR-5582 from the National Institues of Health; and Grant-in-Aid Contract M-43 from the State of New Jersey. Recipient of Research Career Award 5-K3-16,749. from the National Institutes of Health.     

Note on the growth of bacterial populations

In this note the explicit solution is given to an equation, suggested by C. N. Hinshelwood (1946), describing the growth of a bacterial population under the assumption that toxic products are a limiting factor. The behavior of the culture as a function of time and the parameters (initial number, rate of growth, and rate of production of toxic substance) is discussed. Public Health Service Research Fellow of the National Cancer Institute, National Institutes of Health, Federal Security Agency.     

Identification of mouse mammary adipose cells by membrane antigens

Summary Antisera produced to mammary adipose cells from midpregnant BALB/c females can be used to distinguish mammary adipose cells from mammary epithelial cells and fibroblast. The mammary adipose membrane antigen detected by indirect immunofluorescence was found in adipose cells from (a) mammary glands of virgin, midpregnant and lactating mice; (b) mammary fat pads that had been surgically cleared of glandular elements; and (c) epididymis. In all tissues, this cell-surface antigen was removed by the enzymatic action used to dissociate the cells from the tissues and was shown to be fully restored when cells were cultured for 48 hr. This work was supported by National Cancer Institute Grant Nos. CA11736 and CA18946 and Biomedical Research Support Grant No. RR05467 from the National Institutes of Health, DHEW.     

A new RNA synthesis mutant of E. coli

A temperature-sensitive mutant of E. coli is described. At the nonpermissive temperature, the capacity for RNA and protein synthesis decreases logarithmically in the mutant. The mutant is unable to support the growth of f2 or T7 virus, even at the permissive temperature. The temperature-sensitive mutation maps approximately 1 away from rif ^r in E. coli and therefore affects a gene previously undescribed. The temperature sensitivity is suppressed by sublethal concentrations of rifampicin. Moreover, in rif ^r T^s double mutants, the T ^s mutation suppresses rif ^r and vice versa. The partially purified RNA polymerases from mutant and wild-type cells have different temperature and salt optima.This research was supported by Public Health Service grant GM-14368 from the National Institute of General Medical Sciences and by grant IN-29 from the American Cancer Society. One of us (D.P.) is a predoctoral trainee, supported by a National Science Foundation Graduate Traineeship Program and by a National Institutes of Health Predoctoral Research Fellowship. S. Marshall is supported by LASBAU.     

Use of a low-speed,iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations

Summary A simple yet effective method (iso-density percoll centrifugation) has been developed for consistently preparing isolated rat liver parenchymal cells with over 98% initial viability. The method has been applied to cells isolated by a variety of collagenase digestion techniques. This procedure involves the low-speed centrifugation (50 ×g) of the initial cell suspension through a percoll medium having a density of 1.06 g/ml and results in the separation of single and viable parenchymal cells from cell aggregates, debris, and nonparenchymal cells. The enriched parenchymal cells have been shown to be superior to untreated cells by a number of criteria including: preparation homogeneity, cell morphology, maintenance of cytochrome P-450, hormonal responsiveness (measured by the induction of tyrosine aminotransferase after treatment with glucagon or dexamethasone, or both), plasma membrane integrity (determined by both trypan blue exclusion and leakage of glutamic-oxaloacetic transaminase), and the DNA repair capability after treatment with benzo[a]pyrene or 2-acetylaminofluorene. This work was supported in part by the National Institutes of Health Biomedical Research Support Program, and National Institute of Environmental Health Services grant (ES-01737) awarded to M.T.S.H., and by National Cancer Institute grants CA-017175, CA-09135, CA-22484 awarded to H.C.P.N.S. was supported by a Cancer Research Campaign Grant (U. K.) through the International Union Against Cancer. This work was presented in part at the 24th Annual Meeting of the Society of Toxicology, 18–22 March 1985, San Diego, CA.     

Primary culture and mRNA analysis of human ovarian cells

Established cell lines are invaluable for studying cell and molecular biological questions. A variety of human ovarian cancer (OC) cell lines exist, however, most have acquired significant genetic alterations from their cells of origin, including deletion of important cell cycle regulatory genes. In order to analyze signaling events related to cell cycle control in human OC, we have modified existing protocols for isolating and culturing OC cells from patient ascites fluid and normal ovarian surface epithelial (OSE) cells from benign ovarian tissue sections. These cells maintain an epithelial phenotype and can be manipulated experimentally for several passages before cellular senescence. An example using TGF1 treatment of OC cells to examine signaling and target gene activation is presented. This work is supported in part by the National Cancer Institute of Canada with funds from the Canadian Cancer Society (#13631), the Nova Scotia Health Research Foundation, and by Cancer Research and Education (CaRE) Nova Scotia with funding from the Faculty of Medicine, Dalhousie University. L.D.D. is supported by the Rossetti Studentship for Cancer Research with funds from the Dalhousie Medical Research Foundation, T.G.S. is supported by a CaRE Fellowship with funding from the Canadian Cancer Society, and M.W.N is a Canadian Institutes of Health Research Scholar. Published: October 28, 2002.