The lack of v-src involvement in tumorigenicity of marmoset cells transformed in vitro with Rous sarcoma virus.

The transformation of nonhuman primate marmoset cells by Rous sarcoma virus of Schmidt-Ruppin strain (RSV-SR) generates transformants which lack tumorigenicity in allo- and xenogeneic hosts. Marmoset cells acquire this property when they are transformed by RSV rescued from non-tumorigenic allogeneic cells. One of the rescued RSV, when used to infect marmoset kidney cells in vitro, yielded transformants which became tumorigenic in adult allogeneic hosts. Cytogenetic and molecular analyses of transformants revealed progressive genetic changes from cell diploidy to aneuploidy and from the presence to the loss of v-src during propagation in vitro. The loss of v-src in transformed cells coincided with the evolution of aneuploid cell clone with specific marker chromosome (M1). Although both early passage diploid and late passage aneuploid transformants were tumorigenic, the induced tumors originated from different type of cells. Tumors induced by diploid- and v-src-positive transformants were derived predominantly from the host cells, while tumors induced by late-passage transformants and with deleted v-src originated from an aneuploid cell clone that contained a rearranged M1 marker chromosome. These results suggest that besides the v-src oncogene, proviral integration can facilitate chromosomal rearrangements that contribute to tumorigenic transformation of nonhuman primate cells in vitro.

Chronic administration of flumazenil increases life span and protects rats from age-related loss of cognitive functions: a benzodiazepine/GABAergic hypothesis of brain aging.

Under barrier condition and with ad lib access to food and water, 20 Fischer-344 rats were chronically treated for 10 months with the benzodiazepine (BDZ) antagonist, flumazenil (FL; 4 mg/kg/day in drinking water acidified to pH = 3.0), beginning at the age of 13 months, while the group of 20 control age-matched rats received plain acidified water. The life span of the first 8 deceased rats treated with FL was significantly longer than that of the first 8 deceased rats in the age-matched control group. In tests for spontaneous ambulation and exploratory behavior in the Holeboard apparatus, conducted during the 3rd and the 8th month of treatment, the FL group, relative to controls, had significantly higher scores for the ambulation and exploratory behavior. In tests for unrewarded spontaneous alternation in the T maze, conducted at days 7, 39, 42, and 47 through 54 after drug withdrawal, i.e., at the age of 24-25 months, the FL-exposed group, compared to age-matched controls, showed a significantly higher percent of alternating choices, a behavior that was statistically comparable to that of the "young" 6-month-old controls. In the Radial Maze tests conducted 2 months after drug withdrawal, the FL group made significantly less "working memory" errors and "reference memory" errors, relative to the age-matched 25-month-old control group, a performance that was comparable to that of the young 7-month-old control group. In conclusion, chronic FL significantly protected rats from age-related loss of cognitive functions. It is postulated that the age-related alterations in brain function may be attributable to the negative metabolic/trophic influences of the "endogenous" benzodiazepine (BDZ) ligands and/or those ingested with food. A BDZ/GABAergic hypothesis of brain aging has been formulated which assumes that age-related and abnormally strong BDZ/GABAergic influences promote neurodegeneration by suppressing trophic functions of the aminergic and peptidergic neurons through opening of chloride channels in soma membrane and axon terminals, causing excessive hyperpolarizing and depolarizing inhibition, respectively. The review of human clinical and animal data indicates that FL has nootropic actions by enhancing vigilance cognitive and habituation processes.

Phorbol ester promotes growth and transformation of carcinogen-exposed nonhuman primate cells in vitro.

Kidney cells established in vitro from a white-lipped marmoset (106) were exposed to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) alone or in combination with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Low (0.1 micrograms/ml, 4 times), intermediate (1 microgram/ml) and high (1 microgram/ml, 4 times) doses of MNNG resulted in 100%, 50% and 2.8% of cell survival, respectively. High and low doses of MNNG had no effect on cell transformation. Upon exposure of cells to an intermediate dose of MNNG, 106 cells ecquired immortality and evolved into permanent cell line, 106-1M. However, the cells retained normal morphology and anchorage dependence. Chronic applications of TPA (0.1 micrograms/ml, 13 times) promoted 106-1M cells to morphological transformation and anchorage-independent growth but not to tumorigenicity in nude mice (106-1MT cell line). Chromosome analysis revealed only numerical changes in 106 cells and both numerical and structural aberrations in transformed 106-1MT cells. These changes in marmoset cells usually reflected cell culture instability leading to either senescence or to longer survival of cells in vitro. Chronic treatment with TPA did not result in downregulation of protein kinase C (PKC) in transformed 106-1MT cells. Instead, an additional species of PKC appeared in these cells.

Transformation of primate cells by chemicals and oncogenes: requirements for multiple factors.

Although several chemical carcinogenes have been shown to induce malignant transformation in human cells in culture, the molecular events involved in conversion of normal cells to malignant cells remain unknown at present. Normal human cells seem to be resistant to transformation by a single oncogene unless these cells have been immortalized by chemical carcinogens or DNA tumor virus genes. In some human cell systems, malignant phenotype is expressed after activation of protooncogenes probably through the mechanism of point mutations. Our results represent the first induction of KRAS gene rearrangement in cells of patients with familiar polyposis coli (FPC) treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 12-0-tetradecanoyl-phorbol-13-acetate (TPA). Whether or not KRAS rearrangement is a prerequisite for transformation in these cells is unknown. The contrast between the response of the FPC cells following treatment with these chemicals compared with that of normal fibroblasts suggests that KRAS gene rearrangement in FPC cells may be considered as a genetic marker for the disease. Studies of transformation with chemicals and oncogenes extended to nonhuman primate--marmoset cells have shown that two viral oncogenes, v-src and v-sis, converted marmoset cells to altered morphology, anchorage independence and immortality in cell culture. The transformed cells, however, lacked tumorigenicity in allogeneic and xenogeneic hosts. In v-src transformed cells additional changes associated with mutations of transforming virus and chromosome rearrangements were necessary for induction of tumorigenicity in allogeneic marmosets. Transformation of marmoset cells by chemical carcinogens depended on the type of target cells. Skin fibroblasts treated with MNNG acquired transformed morphology, anchorage independence and random chromosome aberrations. Kidney cells treated with the same carcinogen remained untransformed, but attained the same characteristics of transformation when exposed to TPA. Neither skin nor kidney transformed cells showed true tumorigenic potential in nude mice.

Chromosomal and c-K-ras oncogene alterations induced by a chemical carcinogen and phorbol ester in skin fibroblasts of individuals with familial polyposis coli.

Skin fibroblasts derived from three patients with familial polyposis coli (FPC) were treated in vitro with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) alone or in combination with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). None of the cultures treated four times with MNNG alone (1 microgram/ml) or in combination with TPA (eight applications, 0.1 microgram/ml each) showed either morphological transformation or anchorage-independent growth for 18 months after treatments. FPC cells treated with MNNG alone showed cell growth inhibition, breakage and loss of chromosomes, as well as the deletion of 5.7 kilobase (kb) EcoRI fragment in the c-K-ras locus as detected by Southern blot analysis with v-K-ras specific probe (clone KBE-2). The control cells contained two EcoRI fragments of 5.7 and 4.2 kb. On the other hand, cells treated with MNNG first and then followed by treatment with TPA not only showed an increase in the rate of cell growth but also exhibited two novel EcoRI fragments of 9.4 and 12 kb. Treatment with TPA alone appeared to stimulate cell division long after application and to induce chromosomal aberrations but had no effect on c-K-ras sequences. The most likely explanation for the appearance of chromosome pulverization and hyperploidy in FPC cells treated with TPA is the induction of premature chromosome condensation of the S phase cells due to fusion with the mitotic chromosomes.

Instability of v-src sequences in nonhuman primate tumors cultured in vitro.

We have analyzed the DNA of marmoset tumors induced and marmoset cells transformed by Rous sarcoma virus (RSV) and derivative viruses of various types. Southern blot hybridization was used to determine the presence of v-src gene sequences. We failed to detect v-src DNA in high-passage cells derived from marmoset tumors induced in vivo or from marmoset cell lines transformed in vitro. The inability to detect src sequences was not related to selection of revertants in culture, since all cell lines retained transformed morphology and cells transformed in vitro retained the ability to induce sarcomas after transplantation into adult allogeneic marmosets. By contrast, we detected integrated proviruses in cells analyzed 32 to 60 days after in vitro transformation. The proviral sequences appeared to be identical to the transforming virus but were apparently unstable and continued to transpose.

Transplantable primate tumors induced by Rous sarcoma virus. I. Induction of tumors transplantable into young marmosets.

The tumors induced in white-lipped marmosets (Saguinus fuscicollis, S. nigricollis) by Rous sarcoma virus (RSV) of chicken origin (RSV-SR) were not transplantable to allogeneic hosts. In contrast, RSV rescued from these tumors (RSV-M) induced sarcomas that were transplantable to young but not to adult marmosets. The tumors induced by RSV-M and the transplants rapidly enlarged, metastasized to various organs, and killed the recipients 29-59 days post inoculation. Cell lines were readily established from all transplantable sarcomas. No virus expression was detected in transplantable tumor cell lines by electron microscopy or by biochemical and biological assays. However, RSV of the same subgroup as RSV-SR was rescued from both short-term and long-term tumor cell cultures by cocultivation with chicken embryo fibroblasts (CEF). The rescued viruses transformed marmoset cells 100-fold more efficiently than CEF cells, although CEF cells remained permissive for virus replication. Cytogenetic studies revealed extensive chromosome abnormalities in tumor transplants but not in RSV-M-induced sarcomas. All cell lines were hyperploid and contained structurally abnormal, large metacentric and telocentric chromosomes. Immunologic studies failed to detect group-specific (gs) antigen of the avian sarcoma-leukemia complex in either RSV-M-induced, transformed cells or tumor transplants. By complement-dependent cytotoxicity assays, with the use of marmoset anti-gs serum, RSV-associated antigen could be detected on the surfaces of tumor cells. No differences in the expression of this antigen existed between transplantable and nontransplantable marmoset sarcomas. All transplantable cell lines contained abnormal amounts of lipids and glycogen in comparison to RSV-SR-induced tumors and normal marmoset cell lines. The glycogen was associated with unique cytoplasmic membrane complexes and was surrounded by either single- or double-membraned vesicles.

Karyotype of Saguinus labiatus labiatus (red-bellied marmosets).

The karyotype of Saguinus labiatus labiatus was determined by the Giemsa-banding technique on leukocytes cultured from 10 marmosets. The diploid chromosome number (2n = 46) was the same and the chromosome complement similar to other marmosets of genus Saguinus. Small karyotypic differences were found between S. l. labiatus and white-lipped marmosets (Saguinus fuscicollis) in the size of the X chromosome and in the banding pattern of one pair of metacentric chromosomes. A karyotypic variant was detected in 1 S. l. labiatus, characterized by a diploid chromosome number of 45 with balanced autosomal translocation involving two pairs of acrocentric chromosomes (T 16/19).

Syncytium-forming virus of common marmosets (Callithrix jacchus jacchus).

This communication describes the isolation and characterization of a new syncytium-forming virus of common marmosets (Callithrix jacchus jacchus). The virus, isolated from skin explants and peripheral leukocytes of healthy animals, induced syncytia and subsequent cytolysis of several human, simian, and rodent fibroblastic cultures and induced a carrier state in mixed fibroblastic-epithelial or epithelial cell lines. Cytoplasmic and nuclear viral antigen was demonstrated in infected cells by indirect immunofluorescence tests using serum obtained from persistently infected common marmosets. Abundant virus particles were detected within cisternae of endoplasmic reticulum of lytically infected cells by electron microscopy. The virus incorporated [3H]uridine, banded at a density of 1.14 to 1.16 g/cm3 in sucrose, and possessed ribonucleic acid-dependent deoxyribonucleic acid polymerase. No antigenic cross-reactivity was detected between the marmoset virus and simian foamy virus serotypes 1 to 8 in neutralization and immunofluorescence assays. A seroepidemiological survey of a marmoset colony revealed that 53.5% of common marmosets contained antibodies against the virus, whereas other species of marmosets maintained in the same colony remained free of antibodies.

Role of elevated temperature in malignant transformation of mammalian cells in vitro.

Rat embryo cells subjected in vitro to transient incubation at an elevated temperature (39 degrees C) became transformed and induced fibrosarcomas in both homologous and heterologous hosts. Malignant transformation correlated with the occurrence of karyotypic changes which appeared long after incubation at 39 degrees C and subsequent return to 37 degrees C. Control cultures incubated at only 37 degrees C did not show similar chromosomal changes or induce tumors and remained predominantly diploid during the same observation period. In contrast to rat embryo cells, marmoset monkey cell cultures incubated at 39 degrees C did not develop characteristics of transformed cells.

Attempts to transform primate cells in vitro by herpes simplex virus.

Attempts were made to transform human cervical cells and nonhuman primate cells with herpes simplex virus (HSV). None of 129 cervical cell cultures obtained from 29 patients and infected in vitro with various strains of HSV became transformed. Established cell lines of marmoset cells infected with HSV showed no characteristics of malignant transformation. Parallel cultures of rat embryo cells maintained under similar experimental conditions became malignant.

Detection of the viral sarcoma gene product in cells infected with various strains of avian sarcoma virus and of a related protein in uninfected chicken cells.

Genetic analyses have defined a single gene (src) as that portion of the avian sarcoma virus (ASV) genome which encodes the protein directly responsible for ASV-induced neoplastic transformation. We have recently identified the polypeptide product of the src gene of the Schmidt-Ruppin (SR) strain of ASV, a 60,000-dalton phosphoprotein designated pp60(src), and have further determined that pp60(src) acts as a protein kinase. Essential to the identification and characterization of the pp60(src) protein of SR-ASV was the use of serum (TBR serum) from rabbits bearing SR-ASV-induced tumors. TBR serum was, however, strain specific, recognizing pp60(src) from SR-ASV-transformed cells only. We report here that sera from marmosets bearing tumors induced by the Bryan or SR strains of ASV (TBM sera) contain antibody which precipitates the transforming gene product from cells transformed by the SR, Bryan, Prague, or Bratislava strains of ASV. In contrast, rabbits bearing tumors induced by either the Bratislava or Bryan strains of ASV, or hamsters with SR-ASV-induced tumors did not produce antibody to pp60(src) from any strain of ASV. The 60,000-dalton polypeptides immunoprecipitated with TBM serum from cells transformed by each of the above virus strains are phosphoproteins. One-dimensional peptide mapping by limited proteolysis revealed that the pp60(src) proteins are structurally very similar, but not identical. Furthermore, all of the viral pp60(src) proteins have an associated phosphotransferase activity. In addition to detecting the viral src proteins, TBM serum was able to immunoprecipitate an antigenically related protein from normal uninfected avian cells.

Transforming activity of Epstein-Barr virus obtained by superinfection of Raji cells.

Epstein-Barr virus obtained by superinfection of Raji cells with Epstein-Barr virus recovered from P3HR1 cells (HRI virus) transformed human lymphocytes, but it did not superinfect Raji cells. A human lymphoblastoid cell line, HLB, established by such transformation contained 22 Epstein-Barr virus genomes per cell and Epstein-Barr virus-associated nuclear antigen, and a few cells contained early or viral capsid antigen complexes. Chromosomal analysis revealed that HLB-cells were diploid with normal female karyotypes. Replication of Epstein-Barr virus DNA and inhibition of host cell DNA synthesis were observed in HLB cells after superinfection with HR1 virus.

Tumor cell collagenase and its inhibition by a cartilage-derived protease inhibitor.

Human osteosarcoma and mammary carcinoma cells were cultured separately in a medium supplemented with fetal calf serum, until they were confluent. The medium was then replaced by serum-free medium supplemented with heparin. Both cell cultures secreted collagenase, and this activity was inhibited by a cartilage-derived protein of low molecular weight. Since cartilage is rarely invaded by neoplasms, the presence of this inhibitor may play an important role in the regulation of tumor invasion.

Oncogenicity in marmosets of HL-23V, a type C oncornavirus isolated from human leukemic cells, and comparison with simian sarcoma virus type 1 (SSV-1/SSAV-1).

Type C virus produced by dog thymus cells (A7573) that were infected with virus (HL-23V), isolated from cultured leukocytes of an acute myelogenous leukemia patient, transformed marmoset and horse cells in vitro and induced virus-producing fibromas in marmosets. The tumors and transformed foci were indistinguishable morphologically from those induced by simian sarcoma virus, type 1 (SSV-1/SSAV-1). HL-23V was indistinguishable from SSV-1/SSAV-1 by immunofluorescence and neutralization tests, and the nontransforming virus associated with HL-23V completely inhibited SSV-1 focus induction in interference tests. Cell cultures established from a marmoset fibroma produced transforming and nontransforming virus biologically and antigenically indistinguishable from HL-23V and SSV-1/SSAV-1.