Biomedical subjects
Publications and source records attributed to M A Lane.
Blym-1, a transforming gene detected by transfection of NIH 3T3 cells with DNA from Burkitt lymphomas, was mapped to the short arm of chromosome 1 (1p32) by chromosomal in situ hybridization. The Blym-1 gene was not physically linked to the cellular myc oncogene or to any of the immunoglobulin gene loci implicated in the characteristic chromosomal translocations in Burkitt lymphoma.
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A cellular transforming gene detected by transfection of mouse T-cell lymphoma DNA has been isolated by molecular cloning. This gene (designated Tlym-I) is homologous to a small conserved family of sequences present in normal mouse and human DNAs but is not related to any of the previously described viral or cellular transforming genes.
Previous studies identified two glycoproteins of 86 (gp86) and 72 (gp72) kilodaltons and two nonglycosylated proteins of 70 (p70) and 19 (p19) kilodaltons which were specifically expressed in NIH cells transformed by DNA of the MCF-7 human mammary carcinoma cell line. Pulse-chase experiments and the use of tunicamycin to inhibit glycosylation suggested that gp86, gp72, and p19 were related as precursor products. Characteristics of the four transformation-associated proteins resembled those of murine leukemia virus (MuLV) proteins. Sera raised against disrupted MuLV immunoprecipitated the same four proteins in extracts of NIH(MCF-7) cells and MuLV-infected NIH 3T3 cells. In addition, a monoclonal antibody against MuLV gp70 immunoprecipitated proteins gp86 and gp72, whereas a monoclonal antibody against MuLV p15(E) immunoprecipitated gp86 and p19. These results indicate that proteins gp86, gp72, and p19 expressed in NIH(MCF-7) transformants correspond to MuLV envelope proteins gp80env, gp70, and p15(E), respectively. The transformation-associated protein p70 appears to be a non-envelope MuLV protein, most likely p65gag. Northern blot analysis confirmed that transformation of NIH cells by MCF-7 mammary carcinoma DNA led to the induction of an endogenous MuLV provirus.
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DNAs of 20 B- and T-lymphocyte neoplasms of human and mouse origin induced transformation of NIH/3T3 cells with high efficiencies, indicating that these neoplasms contained activated transforming genes that were detectable by transfection. Analysis of the susceptibility of the transforming activities of lymphocyte-neoplasm DNAs to digestion with restriction endonucleases indicated that the same or closely related transforming genes were activated in independent neoplasms representative of the same stage of normal cell differentiation. However, different transforming genes were activated in neoplasms representative of different stages of normal B- and T-lymphocyte differentiation. These results indicate that specific transforming genes are activated in neoplasms of discrete stages of differentiation within these cell lineages.
Sera from tumor-bearing mice immunoprecipitated a 86,000-dalton glycoprotein from extracts of NIH cells transformed by human mammary carcinoma DNA. This antigen was not immunoprecipitated from extracts of NIH 3T3 cells, spontaneously transformed NIH cells, NIH cells transformed by normal human DNA, NIH cells transformed by human bladder carcinoma DNA, or NIH cells transformed by Rous sarcoma virus DNA. In addition, sera from mice bearing tumors induced by NIH cells transformed by either normal human DNA or human bladder carcinoma DNA did not immunoprecipitate this antigen from extracts of NIH cells transformed by human mammary carcinoma DNA. However, this antigen was immunoprecipitated by sera from mice bearing tumors induced by NIH cells transformed by mouse mammary carcinoma DNAs and from mice bearing primary mammary carcinomas. These results indicate that this glycoprotein represents an antigen that is specifically associated with expression of the transmissible transforming genes of human and mouse mammary carcinomas.
High molecular weight DNAs of five tumors induced by mouse mammary tumor virus (MMTV), two mouse mammary tumors induced by a chemical carcinogen, and one human mammary tumor cell line (MCF-7) were assayed for the presence of transmissible activated transforming genes by transfection of NIH 3T3 mouse cells. DNAs of all five MMTV-induced tumors, one chemical carcinogen-induced tumor, and the human tumor cell line induced transformation with high efficiencies (approximately 0.2 transformant per micrograms of DNA). NIH cells transformed by DNAs of MMTV-induced tumors did not contain exogenous MMTV DNA sequences, indicating that MMTV-induced mammary carcinomas contained activated cellular transforming genes that were not linked to viral DNA. The transforming activities of DNAs of all five MMTV-induced tumors, the chemical carcinogen-induced mouse tumor, and the human tumor cell line were inactivated by digestion with the restriction endonucleases Pvu II and Sac I, but not by BamHI, EcoRI, HindIII, Kpn I, or Xho I. These results indicate that the same or closely related transforming genes were activated in six different mouse mammary carcinomas, induced by either MMTV or a chemical carcinogen, and in a human mammary carcinoma cell line.
The potent muscarinic cholinergic antagonist 3-quinuclidinyl benzilate has been used to detect muscarinic acetylcholine receptors on rodent and human lymphocytes. Binding to B lymphocytes was minimal and was not saturable or ligand specific. Half-maximal binding of (3H)-quinuclidinyl benzilate to T lymphocytes occurred at concentrations comparable to those described in other systems, and was displaceable at physiologic concentrations by muscarinic but not nicotinic agonists and antagonists. Binding to T lymphocytes was both saturable and specific, and was found to rapidly increase significantly after mitogen activation. Activation of T lymphocytes or the Lyt 1+ subset produces an early increase and later fall in muscarinic binding, and the ability od cholinergic agonists to augment the effector function of cytotoxic T lymphocytes harvested from alloimmune rat spleen is directly related to the magnitude of muscarinic binding.
BALB/cfC3H female mice (neonatally infected with MTV) possess several different specific antibody activities measured functionally by reactivity with MTV-associated antigens expressed on the surface of isologous MTV-induced mammary tumor target cells. All females when young develop serum antibody which can block specific T-cell cytotoxic activity of BALB/cfC3H spleen cells; this activity remains detectable throughout life. In addition, recruiting antibody is found in the post-weaning stage. This recruiting activity is apparently directed against the same antigenicity as the blocking activity; it can be detected only rarely in whole serum but is revealed in the 19S fraction when the competing blocking antibodies are sequestered into the 7S fraction. In mid-adult life, 19S recruiting activity is found in only a few of the females; the incidence increases as the females age, and it occurs earlier in those females which subsequently develop mammary tumors. A third antibody activity of serum is blocking factor directed against different MTV-associated tumor-cell-surface antigenicity; this blocks the antibody-dependent cell-mediated cytotoxicity characteristic of spleen cells from BALB/c females horizontally exposed to MTV-associated antigenicity. This blocking activity of serum is not found in young females, and in the older females it is more frequent in those which develop mammary tumors.
Our study of the aging process in human beings and in mice is complicated by our need to know whether we are observing diseases of aging or natural nondisease state processes. Results from studies on inbred strains of mice and retrospective studies on HLA types in aging human populations suggest that genetic effects play a significant role in predetermining the life span of an individual. It is clear that in such mouse strains genetic defects that affect cell regulatory mechanisms result in the production of autoimmune reactivity, tumor development, and a shortened life span. In human beings, although results are less clear-cut, strong associations exist between some disease states and the HLA type. Also, the disappearance of HLA-B8 from older women suggests that this HLA type does not confer longevity. Cellular immune reactivity declines with age in all populations studied to date, and cell cooperative or regulatory mechanisms function less well. We need to characterize the specific nature of the cells directly responsible for these alterations and to attempt to correct deficiencies by dietary manipulation or transfer techniques.
Implantation of the mouse mammary tumor virus (MMTV)-producing mammary tumor cell line MJY-alpha into isogeneic mice elicited both humoral and T-cell responses against MMTV virion antigens. The carcinosarcomas which developed from the implanted cells showed a significant decrease in MMTV synthesis, compared with cells remaining in culture, which was detectable as early as 7 days after implantation and for five transplant generations. Electron microscopic examination of thin sections of the tumors revealed that intracytoplasmic A particles, budding particles, and cell-free MMTV B particles were all affected. However, immunofluorescence assays of tumor sections demonstrated the presence of MMTV viral antigens in the cells. Cell cultures initiated from first-, third-, and fourth-generation tumors were morphologically identical to the original in vitro cell line, although virus production was barely detectable. Analysis of the cultures by electron microscopy revealed a significant increase in MMTV virions after in vitro passage 3. Polypeptide profiles obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of virions purified from these cultures were identical to MMTV. Immunodiffusion demonstrated the cross-reactivity between these virions and MMTV particles obtained from mouse milk. In vitro treatment of MJY-alpha cell cultures with rabbit anti-MMTV antiserum resulted in a reduction of extracellular MMTV virions, as well as alterations in their sodium dodecyl sulfate-polyacrylamide gel electrophoretic polypeptide patterns.
Carbamylcholine, acting via a pharmacologically specific receptor, had the ability to activate effector populations of spleen cells from female BALB/cfC3H and BALB/c mice; those cell populations were then significantly reactive in vitro against syngeneic tumor target cells but were only minimally reactive to normal syngeneic target tissues. The induced reactivity was inhibited by the muscarinic cholinergic antagonists atropine, scopolamine, and isopropamide, but not by the nicotinic antagonist d-tubocurare, and it appeared to involve both T-cell and non-T-cell effectors.
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