Suppression of the translocated myc gene and expression of intracisternal A-particle genes in tumorigenic and non-tumorigenic hybrids between murine myeloma and normal fibroblasts

We have studied the tumorigenic potential of a series of independent intraspecies hybrid clones derived from fusion of murine myeloma (BALB/c) and normal fibroblasts (C3H). All of these hybrids grew as adherent cells and thus resembled the fibroblast phenotype. As judged by chromosome enumeration, these hybrids appear to retain the full complement of their parental cells. Three out of 4 hybrids tested were able to form colonies in soft agar and to grow as tumors in either nude or (BALB/c x C3H) F1 mice, albeit at a reduced rate. The 4th hybrid did not grow in agar, was non-tumorigenic and may have had a 2:1 fibroblast to myeloma genomic equivalence ratio. In contrast to the parental myeloma cells, all the hybrids exhibited restricted growth rates in serum-free medium. As in our previous sets of hybrids formed between myeloma and L-cells, expression of the Ig genes was inhibited in the new hybrids and the derived tumors. The constitutive expression of the translocated myc gene in the myeloma parental cells was decreased in the hybrids and in all their derived tumors. In contrast, all of the hybrid cell lines and the tumors express high levels of the intracisternal A particle mRNAs. Our results show that the tumorigenic phenotype of myeloma cells is either fully or partially suppressed in myeloma x fibroblast hybrids and that this may be due to the fact that expression of the translocated c-myc is suppressed. We suggest that, in addition to the translocated myc gene, myeloma cells contain other activated oncogene(s), and that the latter are responsible for the residual tumorigenic potential of the myeloma x fibroblast hybrids.     

Extinction of expression of the translocated myc gene in somatic cell hybrids between mouse myeloma and L-cells

Most murine plasma-cell tumors show a t(12;15) reciprocal chromosomal translocation which truncates the first exon of one of the myc gene alleles and fuses it to one of the switch regions of the immunoglobulin (Ig) heavy-chain locus. This results in constitutive activation of the translocated myc gene and the production of smaller-sized mRNA molecules, which are initiated at new sites in the first myc intron. The normal myc allele is not expressed in these myeloma cells. We have studied the expression of the translocated myc gene in somatic cell hybrids between mouse myeloma and L-cells. Our previous findings show that Ig gene expression is extinguished in such hybrids. In the present work we found that the hybrids contain the normal and translocated myc genes. In contrast to the myeloma parental cells which express the translocated myc gene, the hybrids are similar to the L-cells in expressing only the normal myc allele. Our results suggest that the L-cell, fibroblast-like phenotype, is dominant in these hybrids, and show that the translocated myc gene is expressed in a tissue-specific manner in the context of the myeloma cell, and is not expressed when subjected to a fibroblast-like cellular environment.     

Tumorigenicity of T lymphoma/T lymphoma hybrids and T lymphoma/normal cell hybrids

Stable hybrids formed between clones of established murine T-cell lymphoma lines, and between lymphoma clones and normal spleen or thymus cells were examined for their tumorigenic properties by intravenous (i.v.) and intradermal (i.d.) inoculation into syngeneic AKR mice. Fusion parents consisted of T lymphoma clones of high and low tumorigenicity derived from the SL 12 cell line. In addition, normal spleen cells and thymocytes were fused with poorly tumorigenic T-lymphoma clones. Hybrids tested by i.v. inoculation of 10(6) cells to syngeneic hosts showed that fusion between the lymphoma cells resulted in hybrids which displayed the phenotype of the highly tumorigenic parent. Also, it was shown that fusion of poorly tumorigenic lymphoma cells with normal spleen cells resulted in hybrids with enhanced tumorigenicity. Fusion of poorly tumorigenic lymphoma cells with normal thymocytes resulted in hybrids with the highest tumorigenic potential. The pattern of spread for the tumor/tumor hybrid was that of the highly tumorigenic parent. Tumor spread patterns for the spleen/tumor hybrids were different from those of the thymocyte/tumor hybrids. Intradermal inoculation of 10(5) cells from tumor/spleen or tumor/thymocyte hybrids revealed differences in latent periods between parental and hybrid cells, the tumor/thymocyte hybrids having the shortest latent period. Surface marker studies and T-cell antigen receptor mRNA determinations in the tumor cell/normal cell hybrids indicated that the normal parent was a cell of immature phenotype. Therefore, high tumorigenicity is a dominant characteristic, and poorly tumorigenic but "immortal" T lymphoma cells can derive characteristics which increase their in vivo growth capacity from the putative immature normal cells with which they selectively fuse.     

Immunoregulation of murine and human myeloma.

The studies presented in this review address the issue of immunoregulation of growth and differentiation of myeloma tumor cells by host effector cells (Fig. 1). In the murine myeloma system, substantial, direct evidence is presented that myeloma tumor cell growth and differentiation can be modulated by host idiotype-, antigen-, and isotype-specific immunoregulatory cells. Only isotype-specific immunoregulatory cells, however, represent an endogenous, host-generated response, whereas idiotype- and antigen-specific responses require exogenous manipulation (immunization) of the host. In patients with multiple myeloma, however, little direct evidence is available showing regulation of growth and differentiation of tumor cells. Instead, a substantial body of literature suggests a complex interaction between the host immune system and the tumor that, in principle, may result in regulation of tumor cell growth and/or differentiation, although at best the regulation is clinically ineffective. Changes in the phenotype and functions of T cells, B cells, macrophages, and NK/LAK cells have been demonstrated in patients with multiple myeloma and, at least in vitro, each of these cell populations has been shown to modulate growth/differentiation or survival of human myeloma cells. The future challenge in our studies of the immunobiology of multiple myeloma will be to better understand the mechanisms controlling growth of myeloma tumor cells and the interactions between the tumor cells and the host immune system so that we might develop appropriate strategies to augment antitumor immune responses.     

Gene expression in murine hybrids exhibiting different morphologies and tumorigenic properties

[³⁵S]Methionine labelled polypeptides from mouse CLID, hamsterovary cells (CHO, and 7 derived somatic cell hybrids which havesegregated CHO chromosomes, were analysed by means of high resolutiontwo-dimensional gel electrophoresis under conditions in whichthe position of 600 polypeptides could be reproducibly assessed.As judged from the two-dimensional gel electrophoretic patterns(isoelectric focussing (IEF) and non-equilibrium pH gradientelectrophoresis (NEPHGE)) gene expression in all the hybridsresembled the mouse CLID parent and with only one exceptionthey all expressed different numbers and intensities of CHOspecific polypeptides. Even though some of the hybrids expressedas much as 50% of the total number of CHO specific polypeptidesthat could be clearly differentiated from those of the mouseparent we failed to find a direct correlation between the expressionof any given CHO polypeptide and the morphological or tumorigenicproperties of the hybrids. Most CHO specific polypeptides, however,were expressed at lower levels in the hybrids as compared tothe parent CHO cells, a fact that may be due to the chromosomalconstitution of the hybrids, regulation or both. Similarly,the quantitation of the major cyto-skeletal polypeptides presentin the hybrids, such as a-and B-tubulin, vimentin, total actinand 3 polypeptides (IEF 12, 24 and 31) present in intermediatefilament enriched cytoskeletons, indicated that changes in therelative proportion of any of these proteins is not sufficientto account for the morphology, actin microfilament pattern ortumorigenicity of the hybrids. Co-expression of cytoskeletalproteins in the hybrids could only be demonstrated in the caseof the related mouse IEF 24 and hamster IEF 7 polypeptides.In all other cases the cytoskeletal polypeptides co-migratedand presented similar one-dimensional peptide maps. Some principlesare emerging concerning the possibility of using somatic cellhybridization in combination with two-dimensional gel electrophoresisto locate genes coding for particular polypeptides on a givenchromosome.     

Tumorigenicity and agglutination by concanavalin A of Chinese hamster cells and their hybrids

Two transplantable hamster lymphomas, one of which (ML) metastasizes, while the other (NML) only grows at the site of implantation, were examined in the electron microscope. The tumour cells of the two lymphomas were found to be identical except for a slightly greater amount of ER on ML; both contain the typical ultrastructural features of experimental tumour cells, including viral particles. The difference between the two tumours was found to be in the invading host macrophages. The macrophages in NML are large cells containing phagosomes filled with tumour cells in varying stages of digestion. ML also contains macrophages but these are small cells without phagosomes, which accounts for their not being found in earlier light microscope studies. That these latter cells are macrophages is evident from their membrane processes and the presence of primary lysosomes. It is therefore concluded that the difference between these two lymphomas lies not in the ability of the host macrophages to invade ML, but rather in the inability of the latter macrophages to become activated.     

Tumorigenicity and oncogene expression in pediatric cancers

Cytogenetic and epidemiological studies of pediatric cancers have implicated a loss of genetic information in the development of these tumors. In contrast, other studies have shown that activation of endogenous oncogenes is a common event in these cancer cells. The technique of somatic cell hybridization provides a model for investigating the interaction between loss of genetic elements and oncogene activation in pediatric cancers. A variety of human-human cell hybrids were formed between a tumorigenic adult carcinoma and representative tumorigenic pediatric cell lines. All hybrid cells were completely suppressed for tumor-forming ability when assayed in nu/nu (nude) mice. When the expression of the N-myc, c-myc, and sis oncogenes and tumorigenicity were examined in the same hybrid cells, no correlation was found, suggesting that the expression of these oncogenes in these hybrid cells did not appear to be controlled by putative "tumor suppressor" genes. Thus, tumorigenicity behaves as a recessive genetic trait in pediatric cancers. Furthermore, different genetic elements may be lost during tumor development of adult cancers as opposed to pediatric cancers.     

Tumorigenicity of murine lymphomas selected through fluorescence-detected natural antibody binding

Fluorescence-activated cell sorting was used to isolate high and low IgM natural antibody (NAb) binding populations from a heterogeneous line of the L5178Y-F9 murine lymphoma. The ranking of NAb binding and complement-dependent NAb lysis of the selected and starting lines were the same and opposite to that of their tumorigenicity in syngeneic DBA/2 mice. L5178Y-F9 and SL2-5 clones repeatedly treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate and selected by fluorescence-activated cell sorting for high NAb binding exhibited increases in NAb binding and sensitivity to complement-dependent NAb lysis which corresponded with reduced tumor frequencies of threshold inocula. Although the high NAb binding SL2-5 line was slightly more sensitive to natural killer (NK) cell cytolysis, changes in susceptibility to activated macrophages or hypotonic lysis were not consistent with the observed reductions in tumor frequency so that the selected alterations in NAb binding corresponded best with tumorigenicity. These data confirm the same inverse relationship exhibited previously by in vivo and in vitro selected tumor variants and provide more precise evidence supporting a role for NAb in host resistance against tumor foci.     

Tumorigenicity of SEWA murine cells correlates with degree of c-myc amplification

Previous studies have shown that cells of the SEWA mouse tumor contain amplified copies of the proto-oncogene c-myc in the aberrant chromosomal structures of double minutes (DMs), homogeneously staining regions (HSRs) and C-bandless chromosomes (CMs). DMs, and to a lesser degree CMs, tend to disappear from the cells grown in vitro and again reappear after transfer back in vivo, as if DNA amplification confers a growth advantage upon the tumor cells. We have now isolated five in vitro clones that exhibit different degrees of c-myc amplification. When we inoculated cells of the different clones into compatible hosts, we found that there was a positive correlation between degree of c-myc amplification, level of c-myc RNA, and tumorigenicity. Our results lend further support to the idea that gene amplification contributes to the higher malignant phenotype, and to progression of tumors.     

Inheritance of immunogenicity and metastatic potential in murine cell hybrids from the T-lymphoma ESb08 and normal spleen lymphocytes

T-lymphoma cells were fused with normal lymphoid cells to examine the segregation of tumorigenicity and metastatic capacity in the hybrids. In independent fusions the immunogenic ESb08 T-lymphoma line fused successfully with normal syngeneic spleen cells (from DBA/2 and CD1 mice) enriched either with T-cells or B-cells. Ten times fewer hybrids were obtained with B-cells compared to the number obtained with T-cells, and marker assays showed that both types of fusions preferentially generated T-T hybridomas. Some of the hybrids resembled their tumor parent in their ability to form primary and secondary tumors only in irradiated DBA/2 mice, whereas other hybrids lost the high ESb08 immunogenicity, were equally tumorigenic, and in some cases metastatic, in nonirradiated mice. DNA distributions of the original hybrid lines ranged from a hexaploid DNA content (expected for complete hybrids derived from a tetraploid line and normal diploid cells) to a tetraploid DNA content, confirming the reported chromosome instability of T-T hybrids. No correlation was noted between the initial DNA content and tumorigenicity, but in the case of complete hybrids, reduction in the ploidy levels always was observed in the cells of primary and metastatic lesions. One chromosomally stable and highly malignant hybrid (C2), which was analyzed for segregation of chromosomes and for drug-resistance markers, showed preferential loss of chromosomes from the normal T-cell fusion partner. The decreased immunogenicity of this hybrid could not be related to any detectable loss of chromosomes from the ESb08 tumor parent.     

Argonaute 2与多发性骨髓瘤血管生成临床关系研究

目的 了解Argonaute 2(Ago2)在多发性骨髓瘤(MM)骨髓组织中的表达水平,探讨其与骨髓内血管生成的关系.方法 应用改良的甲基丙烯酸甲酯(MMA)单体塑料包埋法对59例MM患者及16例正常对照进行骨髓活组织检查的组织制片,采用EnVision免疫组织化学二步法检测MM患者及正常对照骨髓组织中Ago2蛋白表达水平及微血管密度(MVD),并用Western blot方法检测8例MM患者和3例正常对照Ago2蛋白表达水平.结果 Western blot检测显示,Ago2蛋白在MM患者骨髓组织中表达高于正常对照(1.35±0.19比0.15±0.03,t=-19.883,P＜0.001).免疫组织化学结果表明,59例MM患者骨髓组织中Ago2蛋白阴性和阳性分别为9例和50例,对照组16例均为阴性,两组差异有统计学意义(x2=42.586,P＜ 0.001).MM患者中,Ago2阳性组β2-微球蛋白高于阴性组,差异有统计学意义(Z=-2.014,P=0.042).MM患者骨髓组织中MVD为7.89±4.88,正常对照组为2.16±1.32,两组之间差异有统计学意义(t=4.63,P＜0.001).MM患者骨髓组织中Ago2蛋白表达水平与MVD具有相关性(r=0.461,P=0.023).Ago2蛋白阳性组MVD较阴性组增高,差异有统计学意义(t=2.71,P=0.009).结论 Ago2在MM患者骨髓组织中表达水平明显升高,可能参与了MM患者的病理发生,并对骨髓内异常新生血管生成起一定的调节作用.     

Tumorigenicity of human HT1080 fibrosarcoma X normal fibroblast hybrids: chromosome dosage dependency

The tumorigenic capacity of hybrids formed by fusion of the highly tumorigenic HT1080 human fibrosarcoma cell line with nontumorigenic normal fibroblasts was examined. The HT1080 also contains an activated N-ras oncogene. Near-tetraploid hybrids which contained an approximately complete chromosomal complement from both parental cells were nontumorigenic when 1 X 10(7) cells were injected s.c. into athymic (nude) mice, whereas the parental HT1080 cells produced tumors in 100% of the animals with no latency period following injection of 2 X 10(6) cells. Tumorigenic variants were obtained from these hybrids which had lost only a few chromosomes compared to cells from the nontumorigenic mass cultures. In addition, several near-hexaploid hybrids were obtained which contained approximately a double chromosomal complement from the HT1080 parental line and a single chromosomal complement from the normal fibroblasts. All of these near-hexaploid hybrids produce tumors in 100% of nude mice with no latency period. Our results indicate that tumorigenicity of these particular human malignant cells of mesenchymal origin can be suppressed when fused with normal diploid fibroblasts. In addition, the results suggest that tumorigenicity in this system is chromosomal dosage dependent, since a diploid chromosomal complement from normal fibroblasts is capable of suppressing the tumorigenicity of a near-diploid but not a near-tetraploid chromosomal complement from the tumorigenic HT1080 parent. Finally, the loss of chromosome 1 (the chromosome to which the N-ras oncogene has been assigned) as well as chromosome 4 was correlated with the reappearance of tumorigenicity in the rare variant populations from otherwise nontumorigenic near-tetraploid hybrid cultures. Our results also suggest the possibility that tumorigenicity in these hybrids may be a gene dosage effect involving the number of activated N-ras genes in the hybrids compared to the gene(s) controlling the suppression of the activated N-ras genes.     

Novel antigen expression on syngeneic somatic cell hybrids of murine spontaneous mammary tumor cells

Spontaneous mammary carcinoma cells of C3H/He mice were fused with syngeneic L-cells, and two types of hybrid cell clones were obtained. In group A, hybrid cells showed contact inhibition, and parental H-2k and L-antigens were well expressed. Metacentric chromosomes of L-cell origin and estrogen dependency were also well preserved in this group. However, in group B, hybrid cells proliferated to pile up, and the expression of parental antigens, H-2 and L-cell antigens, was strongly suppressed. But, mouse mammary tumor antigens (MM-antigens), which were expressed on ascites mammary tumor cells with hypotetraploidy of C3H/He origin and which were not expressed on both parent cells, were newly expressed. The number of metacentric chromosomes was decreased, and estrogen dependency was lost in this group. The relationship between the expression of MM-antigens and that of H-2 antigens was reciprocal, and tumorigenicity was independent of cellular behavior in vitro and of MM-antigen expression. MM-antigen-positive hybrid cell clones were frequently obtained when tumor cells were fused with metaphase-rich L-cells.     

Effects of levamisole on normal and malignant murine lymphocytes.

Levamisole enhanced transformation of murine lymphocytes stimulated either by mitogens or allogeneic lymphocytes. In a similar dose-dependent pattern it stimulated in vitro growth of L1210, P1798, and 6C3HED but not YAC lymphoma cells. Stimulation of growth of lymphoma cells was greater by peritoneal cells harvested from normal mice 4 days after levamisole injection than by peritoneal cells from untreated mice. This effect correlated with the shortened survival time of BALB/c mice treated with levamisole prior to P1798 implantation compared to that of a control group not pretreated. Administration of levamisole with iodoacetamide-modified tumor cells in immunoprophylaxis studies had no effect on the rejection of a tumor implant or on development of tumor-specific antibody. Levamisole was added to regimens involving asparaginase therapy of 6C3HED-bearing C3H mice and chemoimmunotherapy of BALB/c mice bearing P1798 with methotrexate and iodoacetamide-modified P1798 cells. In neither case were there increased numbers of survivors, and mean survival time was generally decreased for the levamisole-treated groups. The stimulated tumor growth may have been mediated by a direct effect of levamisole on the lymphoma cells, through an effect on other cell types, or by both effects; these effects apparently outweighed potentially beneficial effects of levamisole on the immune system.     

Tumorigenicity of friend murine erythroleukemia cell lines differing in spontaneous differentiation rates

The relationship between tumorigenicity and in vitro differentiation was studied in four Friend erythro-leukemia cell lines. The cell lines were isolated by repeated subcloning of an unmutagenized population (sib selection). The cell lines differed in their ability to form tumors when 10³ cells were injected subcutaneously into syngeneic mice (8 to 56% animals with tumors). The ability of cell lines to form tumors was not proportional to their ability to be induced to differentiate by dimethylsulfoxide. The cell lines had indistinguishable chromosome numbers, plating efficiencies, number of cycling cells, population doubling times and spontaneous mutation rates to drug resistance. However, the cell lines had different spontaneous differentiation rates in the absence of dimethylsulfoxide (0.87 to 16.0 × 10^?4 per cell per generation). The ability of cell lines to form tumors was inversely proportional to their spontaneous differentiation rate. Apparently cells which are most efficiently blocked in differentiation have the highest probability of forming tumors.     

Inverse relationship between epidermal growth factor receptor expression and radiocurability of murine carcinomas.

The study investigated whether a relationship exists between the extent of epidermal growth factor receptor (EGFR) expression and in vivo radiocurability of murine tumors. EGFR expression was determined in nine carcinomas (four mammary carcinomas, designated MCa-4, MCa-29, MCa-35, and MCa-K; two squamous cell carcinomas, designated SCC-IV and SCC-VII; an ovarian adenocarcinoma, OCa-I; a hepatocarcinoma, HCa-I; and an adenosquamous carcinoma, ACa-SG) syngeneic to C3Hf/Kam mice using Western blot analysis. These tumors greatly differed in their radioresponse, assessed by TCD50 assay, and in their susceptibility to radiation-induced apoptosis. Likewise, the expression of EGFR greatly varied, by as much as 21-fold, and the magnitude of the EGFR expression positively correlated with increased tumor radioresistance. The levels of EGFR inversely correlated with radiation-induced apoptosis, suggesting that the lack of sensitivity to apoptosis induction was a major mechanism responsible for radioresistance of tumors with high EGFR. This correlation was highly significant only for wild-type p53 carcinomas. Radiation activated EGFR autophosphorylation and increased the activity of protein tyrosine kinase, but only in tumors with high EGFR expression. Thus, EGFR expression was a major determinant of tumor radioresponse in vivo. The pretreatment assessment of EGFR expression could predict radiotherapy outcome and may assist in selecting an effective treatment modality.