A retrovirus-producing transformed mouse cell line derived from a human breast adenocarcinoma transplanted in a nude mouse

Summary A transplantable tumor was established in NIH/Swiss/Nu mice from tissue derived from a human breast adenocarcinoma metastatic to the brain. Cultivation of dispersed cells from the third transplant generation of the tumor produced a rapidly growing, high-density culture of fibroblastlike cells. Chromosome and isozyme assays showed these cells to be of mouse origin. The cells behaved as an established line from initial culture. Cells of the tissue culture line, designated NM-1, produced rapidly growing fibrohistiocytomas in nude mice. Electron microscopy revealed that the cells produced large numbers of type C virus particles. Serological, biochemical, and infectivity assays indicated that the retrovirus produced by NM-1 cells is an ecotropic, infective, murine retrovirus antigenically related to, but distinguishable from, Gross and Moloney viruses. The virus did not transform mouse fibroblasts. The data support the conclusion that mouse stromal cells within the transplanted human tumor had undergone malignant transformation and induction to virus replication. The role of the virus in the malignant transformation remains to be clarified.     

Epithelial and fibroblast cell lines derived from a spontaneous mammary carcinoma in a MMTV/neu transgenic mouse

Female murine mammary tumor virus (MMTV)/neu transgenic mice, expressing a wild-type rat neu oncogene driven by an MMTV promoter, develop focal mammary adenocarcinomas that are pathologically very similar to human breast tumors. Two new cell lines were established from a mammary tumor that arose in a female MMTV/neu transgenic mouse. One of these lines, mammary carcinoma from Neu transgenic mouse A (MCNeuA), has an epithelial morphology, is cytokeratin positive, and expresses high levels of the neu transgene. Karyotyping and comparative genomic hybridization analyses demonstrated genomic alterations in the MCNeuA cell line. The other line, N202Fb3, has a fibroblast morphology, is cytokeratin negative, and expresses the neu transgene at a very low level. This cell line also expresses smooth muscle alpha-actin, suggesting that it is a myofibroblast line. The MCNeuA cell line is tumorigenic when injected into syngeneic MMTV/neu transgenic mice, with an in vivo doubling time of about 14 d. The rationale for establishing this tumor cell line was to provide a tumor transplantation system for rapidly assessing immunotherapeutic interventions before testing in the more cumbersome model of spontaneous tumor development in the MMTV/neu transgenic mice. Mice immunized with a Neu extracellular domain protein vaccine were protected against a subsequent inoculation of MCNeuA cells, indicating that this cell line will be useful for evaluating cancer vaccine strategies. This tumor cell line may also prove useful in studying the biological properties of the neu oncogene and its role in the malignant process. In addition, the tumor-derived fibroblast line may be useful for studying tumor-stromal cell interactions.     

Both T and B cells shed infectious mouse mammary tumor virus.

Mouse mammary tumor virus (MMTV) infected both B and T tissue culture cells and primary B and T cells in vivo after milk-borne transmission of the virus. The infected tissue culture cells processed viral proteins, and both these and primary B and T cells shed virus when cultured in vitro. Moreover, the infected B and T tissue culture cells transmitted virus to uninfected mammary gland cells in vitro. The level of infection of these different cell types in vivo was dependent on the strain of mouse, with C3H/HeN mice showing greater B-cell infection and BALB/c mice greater T-cell infection after nursing on MMTV-infected C3H/HeN mothers. Although their B cells were less infected, BALB/c mice developed tumors more rapidly than C3H/HeN mice. These results indicate that both infected T and B cells are potential carriers of MMTV in vivo.     

Comparative growth of normal and malignant mouse mammary epithelium cultured serum-free on a biomatrix from preadipocytes

Summary Growth of normal and malignant mouse mammary epithelial cells (MMEC) on a biomatrix of substrate-attached material from 3T3-L1 preadipocytes was evaluated to devise culture conditions that are suitable for transformation studies but do not involve embedding cells in a gel. The biomatrix was prepared as described by Levine and Stockdale (18), and serum-free medium contained bovine serum albumin, insulin, progesterone, prolactin, and linoleic acid. Each cell type produced a distinctive pattern of colony architecture in this culture system. Cells from virgin mice (vMMEC) usually formed elaborate, three-dimensional structures resembling ducts and alveoli; cells from pregnant mice (pMMEC) grew as flat monolayers; and tumor cells grew in multilayered clusters. Cell growth was monitored by an assay for succinate dehydrogenase. Similar growth rates were found through Day 8 in cultures of vMMEC and D2 carcinoma cells. Growth of vMMEC slowed thereafter, whereas tumor cells typically continued growing through Day 14 to 18. Increase in cell number during 18 days in culture was 3-, 7-, 9-, and 11-fold for cells from pregnant and virgin mice, BALB/cfC3H and D2 carcinomas, respectively. The percent cells in S phase on Day 2 of culture was 9% for pMMEC, 4 to 11% for BALB/cfC3H tumor cells, 20% for vMMEC, and 24% for D2 tumor cells. Thus, this culture system promotes extended growth of MMEC and offers several advantages over embedding cells in a collagen gel. It may therefore be applicable to in vitro transformation studies with MMEC. Supported by research grant CA-32937 from the National Institutes of Health, Bethesda, MD, to BBA.     

Establishment of hybridomas producing cancer specific human antibodies from B cell line derived from PBL of a patient with adult T cell leukemia

Adult T cell leukemia (ATL) is a malignant disease characterized by tumorous proliferation of CD4⁺ T cells infected with retrovirus human T cell leukemia virus Type-I (HTLV-I) and concurs with an autoimmune disease and cancer due to attenuated immune response. In this study, we established ATL patient derived B-cell line TM-1 producing cancer-specific IgM antibodies, and further characterized its antigen specificity by establishing hybridomas fused with human-mouse origin hetero-myeloma cell line RF-S1. We established three hybridoma cell lines termed 2E12, 3E9, and 3E10, which continuously secreted human IgM antibodies. Immunohistochemical staining of formalin-fixed tissue section using antibodies secreted from these hybridomas showed that these antibodies specifically recognized tumor sites of human colon adenocarcinomas. Antibody produced from hybridoma 3E9 bound to some of leukemic cell lines, but not to normal human PBL, which was evidenced by the flow cytometric analysis, indicating that antibody produced from 3E9 recognizes cell surface antigen specifically expressed in the leukemic cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

A notable phenomenon recapitulated. A fusion product of a murine lymphoma cell and a leukemia virus-neutralizing antibody-producer host plasma cell formed spontaneously and secreting the specific antibody continuously

In the mid-1960s the #620 cell passage line of a murine lymphoma-leukemia was developed at the Section of Clinical Tumor Virology and Immunology, Department of Medicine, The University of Texas M.D. Anderson Hospital in Houston, TX. The diploid lymphoma cells released a retrovirus and were antigenic in young adult Swiss (YAS) mice. Small lymphoma cell inocula were rejected with immunity acquired against large inocula of lymphoma cells. Tissue sections revealed the "starry sky" configurations. In one of the tissue cultures set up from lymphoma #620, a cell line consisting of large round poly- or tetraploid cells arose and was referred to as lymphoma cell line #818. The #818 cells grew in suspension cultures and in the form of large, lethal ascitic tumors in YAS mice. Diploid #620 lymphoma cells stained for retroviral antigens; #818 cells stained both for retroviral antigens and immunoglobulins. Fluids withdrawn from #818 cultures neutralized the leukemia virus in spleen focus assays. Immunoglobulin precipitated from #818 suspension culture fluids strongly and specifically neutralized the leukemia virus. The growth of #620 or #818 cells in YAS mice treated with rabbit anti-lymphoma cell immune sera was strongly inhibited but culture fluids of #818 cells showed weak and insignificant inhibition against leukemia-lymphoma #620 (in one experiment, unpublished). In two experiments #620 lymphoma cells were co-inoculated with immune spleen cells into the peritoneal cavities of YAS mice. The immune spleen cells derived from mice that rejected #620 cell inocula or were actively immunized with a photodynamically inactivated mouse leukemia virus vaccine. In the peritoneal cavities of mice co-inoculated with #620 cells and immune spleen cells, clones of large round cells emerged with tetra- or polyploid chromosomal modes. These cells stained for leukemia viral antigens and immunoglobulins. When passaged in YAS mice these cells induced lethal ascites tumors. It was concluded as early as in 1968-69 that an immune plasma cell can fuse with a lymphoma cell, if the lymphoma cell expresses retroviral antigens against which the plasma cell is producing a specific antibody. Some human lymphoma-leukemia cells express retroviral antigens and/or budding retroviral particles, whether due to the acquisition of new env sequences by incomplete resident endogenous retroviral genomes or due to the entry of exogenous retroviruses into lymphopoietic stem cells. In the Discussion illustrations are provided for the human cell line #778 established from a patient with "lymphosarcoma cell leukemia" in 1966. The malignant cells released unidentified retrovirus-like particles and fused with one another and with reactive lymphoid cells of the host. It should be investigated further if human lymphoma-leukemia cells could fuse with an immune plasma cell of the host and thus alter the clinical course of the disease.     

Selective lethal effect of thymidine on human and mouse tumor cells

Human cell lines derived from a melanoma and a colon carcinoma, and cultures of human melanocytes and intestinal epithelial cells, as well as a mouse mesenchymal non-neoplastic cell line and a malignant subline of the same have been quantitatively studied in tissue culture for their sensitivity to thymidine. All three tumor lines produced solid tumors when injected into nude thymus-deficient mice. No tumors were obtained by injecting cells of the human normal long-term cultures or of the non-neoplastic mouse line. The tumor-producing lines showed a greater sensitivity to the lethal effects of high concentrations of thymidine than their non-tumor-producing counterparts. Less than 23% of the tumor cells survived 72 hours in the presence of 1 mg/ml of thymidine, in contrast to 60% or more of the non-tumor cells. Colony formation was much more inhibited by thymidine and the differential between normal and tumor cells was even more pronounced. Tumor cells which also were treated for 72 hours with 1 mg/ml of thymidine and then plated in fresh medium formed very few colonies. If the plating efficiency of the untreated controls is considered as 100%, 4.3% or less of the treated tumor cells formed colonies, in contrast to 33% or more of the non-tumor cells.     

In vitro culture of various typed meningiomas and characterization of a human malignant meningioma cell line (HKBMM)

We placed on culture the 13 cases of meningiomas, succeeded in making a primary culture of 10 cases and maintained 5 cases in vitro over considerable period of time (over three month), and one cell line derived from a malignant meningioma were established. In the early period of the primary culture, meningioma cells were spindle- or round-shaped cells. In the case of psammomatous type, the cultured cells were characterized as forming psammoma bodies. A cell line designated "HKBMM" was established from a human malignant meningioma occurred from frontal lobe. This line grew well without interruption for 5 years and was subcultivated over 120 times. The cells were spindle and fibrous in shape, and neoplastic and pleomorphic features, and multilayering without contact inhibition. The cells proliferated rapidly, and the population doubling time was about 29 hours. The chromosome number showed a wide distribution of aneuploidy. The mode was in the diploid range. The culture cells were easily transplanted into the subcutis of nude mice and produced the tumor resembling the original tumor.     

Biological properties of a type C virus isolated from a human X mouse hybrid cell line.

The biological properties of the HMV-1 virus, spontaneously released from a human X C57BL/6 mouse hybrid cell line, were similar to those of RadLV, the prototype B-tropic virus of C57BL/6 mice. Both viruses replicated on B-type mouse cells and in the wild mouse cell line SC-1. The plaque-forming abilities of the two viruses were relatively low, but gradually increased after passage in new host cells. Both viruses were neutralized by AKR antisera but not by FMR antisera. HMV-1 virus could rescue the defective sarcoma genome from S+H- mouse cells. The pseudotype sarcoma virus so produced was deficient in "helper virus" activity. Newborn mice inoculated with HMV-1 virus remained tumor-free over a 1-yr observation period.     

带有逆转录病毒的恶性T淋巴细胞株的建立

从一例患神经系统疾病病人的外周血淋巴细胞中建立了一株恶性Ｔ淋巴细胞株ＣＭ－１并研究了它的生物学特性。用过滤的ＣＭ－１细胞的培养上清,可命名多发性血管硬化症病人的淋巴细胞转化恶性Ｔ淋巴细胞,由此建立ＣＭ－２细胞株。用ＣＭ－１和ＣＭ－２细胞皮下接种裸鼠,都能使裸鼠产生弥漫性恶性淋巴瘤。电镜下见到了类似于Ｃ型逆转录病毒的颗粒,逆转酶活性检测阳性。血清学和基因检测表明ＣＭ－１和ＣＭ－２中不存在本室常用的其     

Activation of retrovirus in transgenic mice: association with development of olfactory neuroblastoma.

A line of transgenic mice that express the human adenovirus type 12 E1A and E1B genes under the regulatory control of the mouse mammary tumor virus long terminal repeat was studied. Mice from this line develop olfactory neuroblastomas at approximately 6 months of age. Large numbers of type C retrovirus (ecotropic murine leukemia virus) particles were found in the tumor rosettes. No similar examples of virus activation were identified in tumors from other transgenic experiments. Examination of spontaneous olfactory neuroblastomas from three domestic cats also demonstrated retrovirus in tumor rosettes.     

Accelerated malignant conversion of human HBL-100 cells by the v-Ki-ras oncogene

The human epithelial HBL-100 cell line harbors SV40 genetic information and has an unlimited growth potential. Despite displaying properties characteristic of transformation since its early in vitro passages, it is capable of producing progressively growing tumors in nude mice only after long-term culture. This is a reproducible phenomenon and apparently not the consequence of a selection of preexisting malignant cells. Superinfection of early passage nontumorigenic HBL-100 cells with Kirsten murine sarcoma virus, which contains a Ki-ras oncogene having undergone multiple activating events, induces morphologic alterations and rapidly converts the cells to neoplastic cells, further supporting the hypothesis of multistep carcinogenesis. The HBL-100 cell line might be useful in defining the oncogenes representative of different families, which are able to complement SV40 in this system.     

mMaspin: the mouse homolog of a human tumor suppressor gene inhibits mammary tumor invasion and motility.

BACKGROUND: The human maspin gene encodes a protein in the serine proteinase inhibitor (serpin) family with tumor-suppressing functions in cell culture and in nude mice. In order to examine the role of maspin in an intact mammal, we cloned and sequenced the cDNA of mouse maspin. The recombinant protein was produced and its activity in cell culture was assessed. MATERIALS AND METHODS: Mouse maspin (mMaspin) was cloned by screening a mouse mammary gland cDNA library with the human maspin cDNA probe. Northern blot analysis was used to examine the expression patterns in mouse tissues, mammary epithelial cells, and carcinomas. Recombinant mMaspin protein was produced in E. coli. Invasion and motility assays were used to assess the biological function of mMaspin. RESULTS: mMaspin is 89% homologous with human maspin at the amino acid level. Like its human homolog, mMaspin is expressed in normal mouse mammary epithelial cells and down-regulated in mouse breast tumor cell lines. The expression is altered at different developmental stages in mammary gland. Addition of the recombinant mMaspin protein to mouse tumor cells was shown to inhibit invasion in a dose-dependent manner. As with the human protein, recombinant mMaspin protein also inhibited mouse mammary tumor motility. Deletion in the putative mMaspin reactive site loop (RSL) region resulted in the loss of its inhibitory functions. CONCLUSIONS: mMaspin is the mouse homolog of a human tumor suppressor gene. The expression of mMaspin is down-regulated in tumor cells and is altered at different developmental stages of mammary gland. mMaspin has inhibitory properties similar to those of human maspin in cell culture, suggesting that the homologous proteins play similar physiological roles in vivo.     

Production of the mouse mammary tumor virus in cultures of BALB/cfC3H strain

The mouse mammary tumor virus (MTV) reproduces by a budding mechanism at the cell membrane of mouse mammary epithelial cells. In tissue culture, the tumor cells release their virions in the culture supernatant from which they can be removed by high speed centrifugation. Mammary tumor cells from the RIII, GR, and A strains of mice generally produce yields of virus which decrease after a few months. Cells derived from a spontaneous mammary tumor in a BALB/cfC3H mouse have shown the capability to shed relatively large amounts of virus continuously. A quantitative estimation by membrane immunofluorescence of the number of virus producing cells in one-year-old cultures revealed the presence of viral antigen on 80 to 90% of the cells; by comparison, cultures from other mouse strains had a ratio of only 10 to 15% virus producing cells. High speed centrifugation pellets obtained from 50 ml culture supernatant provided large amounts of mature virus particles which have been characterized by electron microscopy.     

A retrovirus vector expressing the putative mammary oncogene int-1 causes partial transformation of a mammary epithelial cell line

In mammary tumors induced by the mouse mammary tumor virus (MMTV), the int-1 gene is frequently activated by adjacent proviral insertions and is thereby strongly implicated in tumorigenesis. To seek a direct biological effect of int-1 that would validate its proposed role as an oncogene, we constructed a retrovirus vector containing the gene and examined its effects on tissue culture cells. Expression of int-1 in a mammary epithelial cell line caused striking morphological changes, unrestricted growth at high cell density, and focus formation on a monolayer, although the cells were not tumorigenic in vivo. This partial transformation induced by int-1 was not observed in cells infected by an otherwise identical virus bearing a frameshift mutation in the gene. These findings strongly support the hypothesis that int-1 plays a functional role in MMTV-induced mammary tumorigenesis.     

In Vitro and In Vivo Observations on a Murine C-Type Virus

From 40 discrete mouse tissue culture cell lines examined by electron microscopy or complement fixation, or both, for the presence of detectable virus, one (NCTC 4705), initiated and maintained on chemically defined medium, was chosen for a more extensive study. Virus-like particles (100 to 110 mμ), morphologically similar to previously reported immature and mature C-type leukemia virus particles, were found budding from the plasma membrane and free in the intracellular spaces of cells in tissue culture and in fibrosarcomas resulting from intramuscular implants of these tissue cultures. Complement-fixation tests for group reactive murine leukemia antigens were positive, with titers consistently higher to a broadly reactive anti-serum than to anti-Friend, anti-Moloney, or anti-Rauscher sera. The 4705 virus was neutralized by Gross antiserum, but not by the F-M-R antisera. When injected into DD, BALB/c, or C3H/He newborn mice, the virus thus far has manifested no leukemogenicity, though virus from tumor extracts and tissue culture medium has been shown to be capable of infecting C3H and Swiss mouse embryo tissue cultures and successfully replicating in them. The role of the virus in accelerating or inducing neoplastic transformation in NCTC 4705 is still not known. When it was introduced into NCTC [ill], a non-neoplastic cell line in other respects similar to NCTC 4705, 4823 manifested no signs of neoplastic transformation after harboring the virus more than 300 days in vitro.     

Role of virus receptor Hyal2 in oncogenic transformation of rodent fibroblasts by sheep betaretrovirus env proteins

The ovine betaretroviruses jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) cause contagious cancers in the lungs and upper airways of sheep and goats. Oncogenic transformation assays using mouse and rat fibroblasts have localized the transforming activity to the Env proteins encoded by these viruses, which require the putative lung and breast cancer tumor suppressor hyaluronidase 2 (Hyal2) to promote virus entry into cells. These results suggested the hypothesis that the JSRV and ENTV Env proteins cause cancer by inhibiting the tumor suppressor activity of Hyal2. Consistent with this hypothesis, we show that human Hyal2 and other Hyal2 orthologs that can promote virus entry, including rat Hyal2, can suppress transformation by the Env proteins of JSRV and ENTV. Furthermore, we provide direct evidence for binding of the surface (SU) region of JSRV Env to human and rat Hyal2. However, mouse Hyal2 did not mediate entry of virions bearing JSRV or ENTV Env proteins, bound JSRV SU poorly if at all, and did not suppress transformation by the JSRV or ENTV Env proteins, indicating that mouse Hyal2 plays no role in transformation of mouse fibroblasts and that the Env proteins can transform at least some cells by a Hyal2-independent mechanism. Expression of human Hyal2 in mouse cells expressing JSRV Env caused a marked reduction in Env protein levels, indicating that human Hyal2 suppresses Env-mediated transformation in mouse cells by increasing Env degradation rather than by exerting a more general Env-independent tumor suppressor activity.     

Restoration of wild-type p53 in drug-resistant mouse breast cancer cells leads to differential gene expression, but is not sufficient to overcome the malignant phenotype

We established a breast cancer cell line from a fast growing mouse WAP-SVT/t breast tumor. Cells from this line, SVTneg2, switched off T-antigen expression, carry a missense mutation at the p53 codon 242 (mouse G242 corresponds to human hot spot mutation G245), are malignantly transformed, highly aneuploid and very insensitive to apoptotic stimuli. To examine the influence of wild-type p53 (wtp53) restoration on the behavior of the SVTneg2 cells, we transfected these cells with wtp53 and generated three permanent cell lines expressing wtp53. Interestingly, restoration of p53 had no influence on chemotherapy sensitivity and the transformation capacity of these breast cancer cells, but markedly changed the gene expression of wtp53-dependent genes after doxorubicin treatment. We postulate that restoration of p53 leads to massive changes in gene expression and to a reduced proliferation rate, but is not sufficient to overcome the malignant phenotype and the chemoresistance of SVTneg2.