Analysis of MOPC-315 plasmacytoma by elutriation and flow cytometry

Intravenously transplanted murine plasmacytoma MOPC-315 cells were separated from normal spleen cells from a tumour-bearing mouse by elutriation and characterized according to morphology, immunologic properties and clonogenicity. Morphologically, both lymphocytoid and plasmacytoid cells were separable by elutriation. Flow cytometry correlated DNA content and intracytoplasmic IgA content and demonstrated two distinct populations, both in cell cycle, but with markedly different cellular IgA levels. Density gradient separation characterized the lower-density cells with lower IgA content and higher clonogenicity. From these studies a model of cellular differentiation is proposed.     

The murine IgA-secreting plasmacytoma MOPC-315 expresses somatostatin receptors

We have previously shown that some neuropeptides had a profound effect on in vitro Ig synthesis (especially IgA) and mitogen-driven murine lymphocyte proliferation. MOPC-315, an IgA-secreting plasmacytoma line, has been extensively used in studies of the regulation of IgA synthesis. In this report we show that the neuropeptide somatostatin (SOM) inhibits proliferation ([3H]thymidine uptake) of MOPC-315 and also inhibits IgA synthesis in vitro. MOPC-315 cells bind both fluorescent SOM and [125I]SOM specifically. On cytofluorimetric analysis, 68 +/- 6.8% (mean +/- SE, n = 7) of MOPC 315 cells labeled with fluorescent SOM and this staining was compatible by incubation with an excess of unlabeled peptide. Specific [125I]SOM binding increased linearly with cell concentration, was rapid and achieved equilibrium after 20 min at 4 degrees C. It was temperature-dependent, readily reversible, and under equilibrium conditions demonstrated a dissociation constant of 1.6 +/- 0.7 nM (mean +/- SE, n = 5). Scatchard analysis showed that MOPC-315 cells had 40,733 +/- 16,050 (mean +/- SE) binding sites for SOM per cell. The characteristics of the interactions of SOM with MOPC-315 cells suggest a specific receptor-mediated mechanism whereby this neuropeptide may modulate lymphocyte function.     

Precursor sequence of MOPC-315 mouse immunoglobulin heavy and light chains.

Partially purified mRNA coding for the MOPC-315 heavy (alpha) or light (lambda 2) immunoglobulin chain was translated in a nuclease-treated reticulocyte lysate containing 20 labeled amino acids. Radiolabeled precursor heavy and light chains, purified by immunoprecipitation and preparative gel electrophoresis, were subjected to Edman degradation. The labeled phenylthiohydantoin derivatives obtained in each degradative cycle were identified and quantitated by high pressure liquid chromatography. Both heavy and light chain precursor segments were hydrophobic in nature; however, they were not homolgous in sequence. To establish whether COOH-terminal proteolytic processing of the heavy chain might also be occurring during secretion, the cyanogen bromide peptides of the heavy chain precursor were compared to those of the mature secreted heavy chain. The results indicated that the COOH termini of the two chains were identical.     

Messenger RNA for immunoglobulin light and heavy chains in MOPC-315 plasmacytoma and variants

Mouse plasmacytoma MOPC-315 produces light and heavy immunoglobulin chains. The variants, MOPC-315 NP-1 and MOPC-315 NR, synthesize only heavy or light chains, respectively. To eludicate the inability of MOPC-315 variants to produce intact light or heavy chains, complementary DNAs (cDNAs) to the mRNAs were prepared. From the kinetics of DNA-RNA hybridization, the RNA of the MOPC-315 NP-1 variant was shown to contain few or no sequences homologous with MOPC-315 light chain mRNA. Thus, the inability of this variant to produce light chain results from the absence of light chain mRNA. In contrast, the polyadenylated mRNA fraction of the MOPC-315 NR variant, which does not synthesize heavy chain, contains about two-thirds of the sequences present in heavy chain mRNA. Thus, this variant contains a fragment of heavy chain mRNA.     

Idiotype-specific transplantation resistance to MOPC-315: abrogation by post-immunization thymectomy.

The idiotype-specific myeloma transplantation resistance induced in BALB/c mice by immunization with the DNP-binding IgAlambda2 protein produced by plasmacytoma MOPC-315 is ablated by post-immunization thymectomy. Sham-thymectomy has no effect. The ablative effect of thymectomy is observed is observed in mice challenged subcutaneously with MOPC-315 cells either 3 days after thymectomy, or after a rest period of 44 days after thymectomy. These observations suggest that short-lived, thymic-dependent suppressive factors may play a role in the idiotype-specific myeloma graft resistance.     

Isolation and properties of poly(A)-mRNA from mouse plasmacytoma MOPC-21

The non-degradable immunoglobulin polyribosomes were isolated from mouse plasmocytoma MORS-21 according to a previously developed immunochemical procedure. The physico-chemical and biological properties of poly (A)-mRNA isolated from individual polyribosomes were studied. The mRNA preparations obtained can be used as a model for studies of current problems of gene engineering.     

Phospholipid methylation in MOPC-31C cell membranes with modified phospholipid composition

The presence of the methylation pathway from phosphatidylethanolamine to phosphatidylcholine was first shown in MOPC-31C cells. Intermediate phospholipids of this pathway, phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N′-dimethylethanolamine, were accumulated in the cell membranes by adding choline analogues such as N-monomethylethanolamine and N,N′-dimethylethanolamine to the culture medium. These modified membranes had a striking character of enhanced phospholipid methylation. This enhancement could be explained by increases in the second and the third step of the methylation pathway from phosphatidylethanolamine.     

Synthesis and processing of the mouse MOPC-321 kappa chain in Xenopus laevis oocytes.

Polyadenylated mRNA isolated from mineral oil-induced plasmacytoma (MOPC)-321 was injected into Xenopus laevis oocytes that were incubated in ³H-labeled amino acids. The MOPC-321 k chain was purified from an oocyte homogenate by immunoprecipitation, followed by preparative gel electrophoresis. To determine whether the precursor segment had been properly and precisely cleaved in the oocyte, the amino acid sequence of the NH₂terminal end of the purified k chain was investigated. The NH₂-terminal sequence obtained was identical to that of the mature, secreted form of the protein. Thus the specificity of the enzyme performing the cleavage of precursor to mature chain is similar in frog oocytes and in mammalian cells. Therefore, the enzymatic specificity has been highly conserved during evolution and evidently performs an essential role in cellular metabolism.     

Presence of an enlarged pool of MOPC-315-specific cytotoxic T lymphocyte precursors in the thymuses of mice that eradicated a large MOPC-315 tumor as a consequence of low-dose melphalan therapy

Summary We have previously shown that, as a consequence of low-dose melphalan (l-phenylalanine mustard) treatment, thymocytes from mice bearing a large, day-10 MOPC-315 tumor, but not thymocytes from normal mice, acquire the ability to generate an enhanced level of antitumor cytotoxicity upon in vitro stimulation with MOPC-315 tumor cells plus low concentrations (9.0–90 IU/ml) of exogenous interleukin-2 (IL-2) [23]. Here we show that the time interval between tumor inoculation and low-dose melphalan therapy as well as the magnitude of tumor burden at the time of the chemotherapy are important for the ability of the drug to render thymocytes more responsive to in vitro stimulation with MOPC-315 tumor cells plus low concentrations of recombinant IL-2 (rIL-2). Specifically, the chemotherapy was found to be effective in enhancing the thymic antitumor reactivity only if the mice bore a large, late-stage tumor. Comparison of thymocytes from untreated mice bearing a large, late-stage tumor to thymocytes from normal mice revealed that tumor-bearer thymocytes contained approximately a three-fold higher frequency of cytotoxic T lymphocyte precursors (CTLp) for MOPC-315-associated antigens. Following curative low-dose melphalan therapy of mice bearing a large, latestage MOPC-315 tumor, the frequency of CTLp for MOPC-315-associated antigens increased further, reaching a level approximately tenfold higher than that found among thymocytes of normal mice. At the same time, the frequency of CTLp for an antigenically unrelated allogeneic tumor (EL4) as well as the overall percentage of mature cells was not increased. The cells responsible for the exertion of the enhanced antitumor cytotoxicity following in vitro stimulation of thymocytes from mice treated with low-dose melphalan when they have a large, latestage MOPC-315 tumor are of the CD8⁺/CD4^– phenotype. Thus, the enhanced level of antitumor cytotoxicity generated by thymocytes from mice that are treated with low-dose melphalan when they have a large, late-stage MOPC-315 tumor is due, at least in part, to the presence of an enlarged pool of CTLp specific for MOPC-315-associated antigens, which mature into CD8⁺/CD4^– effector cells upon stimulation with MOPC-315 tumor cells plus low concentrations of rIL-2.supported by Research Grant CA-35761 from the National Cancer Institute and Research Grant EY-05945 from the National Eye Institute. This work is in partial fulfillment of the requirements for the Doctor of Philosophy degree of M. M. B. M. B. M. was supported by a Career Development Award CA-01350 from the National Cancer Institute.     

Deficiency in immunocompetence of mice cured from large MOPC-315 plasmacytomas by melphalan therapy

Summary Mice cured from large MOPC-315 tumors by a single dose of melphalan, 7.5 mg/kg, were examined for up to 60 days after the drug treatment (71 days after the tumor inoculation) for their ability to respond to mitogenic stimulation, specific and nonspecific antigenic stimulation and for their susceptibility to inoculation with an unrelated tumor, L10 lymphoma. The response of spleen cells from cured mice to mitogenic stimulation by phytohemagglutinin or concanavalin A was slightly depressed at an early stage after the drug treatment. The allogeneic response against C57BL spleen cells and the antibody response against sheep red blood cells (SRBC) of spleen cells from cured mice remained below normal levels during the whole observation period. The deficiency in response to antigenic stimulation was found to be due to impairment in T-cell function. Cured mice were also deficient in their response to SRBC immunization (antibody and delayed-type hypersensitivity responses) and were more susceptible to inoculation with an unrelated tumor, L10 lymphoma, than normal, noninoculated mice. On the other hand, spleen cells of cured mice developed a highly specific cytotoxic response against target MOPC-315 tumor cells and the cured mice were resistant to challenge with an otherwise highly tumorigenic dose of MOPC-315. Thus, cured mice remained deficient for a long period of time in their response to MOPC-315-unrelated antigens but, at the same time, they showed a potent specific antitumor immunity potential in vivo and in vitro.Presented in part at the Ninth European Immunology Meeting, September 14–17, 1988, Rome, ItalyThe contribution of S. Shoval is in partial fullfillment of a PhD Thesis     

Asymmetrical surface IgG on MOPC-21 plasmacytoma cells contains membrane heavy chain and one secretory heavy chain

Membrane proteins from the B lymphomas WEHI-231 and 2PK3 and from the plasmacytomas MPC-11 and MOPC-21 were radioiodinated in situ by the lactoperoxidase method and were subjected to two-dimensional (nonreduced, reduced) polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Few heavily labeled membrane proteins were composed of disulfide-bonded subunits. One such protein (m.w. 200,000 intact and 116,000 reduced) shared some properties with the PC-1 alloantigen, although it was not conclusively identified. A second major disulfide-bonded protein (m.w. 200,000 intact and 95,000 reduced) has been identified previously as the receptor for transferrin. Membrane immunoglobulins of WEHI-231 (IgM) and 2PK3 (IgG2a) had the expected subunit structure, whereas membrane immunoglobulin was not detected on MPC-11. In contrast, surface IgG1 of MOPC-21 appeared to consist almost entirely of hybrid molecules containing one membrane gamma 1 chain and one secretory gamma 1 chain. This hybrid IgG molecule appeared to exist in both monomeric and dimeric forms. It is concluded that i) the synthetic and assembly mechanisms of secretory and membrane IgG1 are shared; ii) there are no special mechanisms to prevent pairing of membrane and secretory gamma 1 chains; iii) the presence of one hydrophobic tail is sufficient for membrane insertion of gamma 1 chains; and iv) the C-terminal extension cysteine residues of membrane gamma 1 chains in hybrid IgG molecules are either unpaired or may allow the formation of hybrid IgG dimers.     

Eradication of a large MOPC-315 tumor in athymic nude mice by chemoimmunotherapy with Lyt2+ splenic T cells from melphalan-treated BALB/c mice bearing a large MOPC-315 tumor

Summary We have previously shown that spleen cells from BALB/c mice that are in the process of eradicating a large MOPC-315 tumor following low-dose (2.5 mg/kg) melphalan (l-phenylalanine mustard) therapy are effective in preventing tumor progression upon adoptive transfer into BALB/c mice bearing a barely palpable tumor that had been treated with a subcurative dose of melphalan [Mokyr et al. (1989) Cancer Res 49: 4597]. Here we show that such spleen cells in conjunction with a subcurative dose of drug (adoptive chemoimmunotherapy, ACIT) can cause the complete regression of a large (15–20 mm) s.c. MOPC-315 tumor in a large percentage of T-cell-deficient (athymic nude) tumor-bearing mice. Spleen cells that were effective in ACIT of athymic nude mice displayed in vitro a substantial direct lytic activity against MOPC-315 tumor cells, and the lytic activity was greatly enhanced when the spleen cells were cultured for 5 days with or without mitomycin-C-treated MOPC-315 stimulator tumor cells. The cells responsible for the therapeutic effectiveness of the spleen cells in ACIT of athymic nude mice, as well as the cells responsible for the direct in vitro anti-MOPC-315 lytic activity of the spleen cells, were of the Lyt 2 and not the L3T4 phenotype. Most of the athymic nude mice that completely eradicated a large MOPC-315 tumor as a consequence of ACIT were capable of rejecting a challenge with 30–100 times the minimal lethal tumor dose for 100% of normal BALB/c mice administered more than 1 month after the ACIT. The ability of these athymic nude mice to resist the tumor challenge was associated with the presence of a greatly elevated percentage of cells expressing T cell surface markers in their spleens. Thus, it is conceivable that splenic Lyt 2⁺ T cells from melphalan-treated BALB/c mice bearing a large MOPC-315 tumor mediate their therapeutic effectiveness in ACIT of athymic nude mice bearing a large MOPC-315 tumor, at least in part, through direct cytotoxicity for MOPC-315 tumor cells. In addition, eradication of a large MOPC-315 tumor through cooperation between antitumor immunity and melphalan toxicity endues the athymic nude mice with an elevated percentage of T cells in their secondary lymphoid organs, and these T cells are probably responsible for the long-lasting protective antitumor immunity exhibited by these mice.Supported by research grant IM-435A from the American Cancer Society and research grant B-8806 from the Bane EstateIn partial fulfillment of the requirements for the Doctor of Philosophy DegreeRecipient of Career Development Award CA-01350 from the National Cancer Institute     

Characterization of infectious oncornaviruses from MOPC-460 plasmacytomas: their relation to A-type particles.

MOPC-460 mouse plasmacytoma cells produce intracellular A-type particles and extracellular oncornavirus-like particles ("myeloma-associated virus," abbreviated MAV). The genomes of these two particles are closely related. During attempts to establish infections with MOPC-460 extracellular particles, we isolated ecotropic and xenotropic infectious forms of murine leukemia virus. We have investigated the relation of these isolates to A-type particles and to MAV by nucleic acid hybridization. Using complementary DNA probes prepared from the two isolates, we found that these infectious murine leukemia viruses differ from A-type particles and from MAV. Moreover, we found that MAV is the predominant extracellular component: the ecotropic and xenotropic forms of murine leukemia virus were present at only low levels (less than 5%) in MAV preparations. Neither the SC-1 cells infected with ectropic murine leukemia virus nor the mink cells infected with xenotropic murine leukemia virus showed any A-type particles in their cytoplasm when examined by electron microscopy. Our inability to demonstrate infection by the A-type particle-related component, MAV, suggests that these may be defective.     

Synthesis and glycosylation of the MOPC-46B immunoglobulin in kappa chain in Xenopus laevis oocytes.

Polyadenylated mRNA isolated from MOPC-46B plasmacytoma, which secretes a glycosylated kappa chain, was injected into $\text{Xenopus laevis}$ oocytes. Analysis of the resulting product showed that [1-¹⁴C]mannose was incorporated into the MOPC-46B kappa chain. Light chains synthesized in oocytes injected with mRNA from MOPC-321 plasmacytoma, which secretes a nonglycosylated kappa chain, failed to incorporate label from [1-¹⁴C]mannose. Thus, protein glycosylation in the oocyte is apparently specific in that carbohydrate is incorporated only into the kappa chain synthesized as a glycoprotein by myeloma cells. It is thus evident that the general signals for glycosylation have remained stable during independent evolution of the amphibia and mammalia.     

Relationships between intracisternal type A and extracellular oncornavirus-like particles produced in murine MOPC-460 myeloma cells.

Oncornavirus-like particles of the "A" (both intracisternal and intracytoplasmic) and "B" or "C" (extracellular) types are produced by murine MOPC-460 myeloma cells. This communication describes a comparative study on tracisternal A and extracellular particles. Both types of particles contain an RNA-dependent DNA polymerase activity, traces of 35S and 70 S RNA in addition to larger amounts of degraded RNA, and proteins of approximately 76,000 and 45, 000 daltons. The 76,000-dalton proteins from intracisternal A and extracellular particles have the same cyanogen bromide peptides. Hybridization kinetic analysis indicates that the RNAs in the two particles are identical or very closely related and share partial homology with Moloney leukemia virus RNA. In contrast, the particles appear to have little or no relationship to murine mammary tumor virus as judged by several different criteria. Electron microscope studies indicate that the extracellular particles arise from the budding of core components through the plasma membrane. These results suggest that the intracisternal A and extracellular oncornavirus-like particles produced by MOPC-460 cells are closely related.     

De novo synthesis and glycosylation of the MOPC-46B mouse immunoglobulin light chain in cell-free extracts.

The mRNA for the mouse MOPC-46B K chain, a glycorportein, was prepared translated in Ehrlich ascites cell-free extract. A polypeptide product similar to the MOPC-46B kappa chain was identified by its antigenic properties and by analysis of the products of tryptic digestion. In addition, the cell-free product was glycosylated after translation. It appeared that glycosylation of the cell-free product occurred on the same tryptic peptide glycosylated by intact cells. The characteristics and requirements of glycosylation are described. The presence of Mn2+ was a major requirement.