Alteration of the metastatic potential of line 1 lung carcinoma cells: opposite effects of class I antigen induction by interferons versus DMSO or gene transfection.

Class I antigens are necessary for the recognition of tumor cells by cytotoxic T lymphocytes (CTL). The line 1 lung carcinoma is a spontaneous murine tumor deficient in class I antigen expression. Consistent with this, line 1 cells are highly metastatic in vivo. We investigated whether increasing class I antigen expression on line 1 cells could alter the metastatic potential of these tumor cells using an in vivo lung metastasis model. We used three methods to induce class I antigen expression on line 1 cells: gene transfection, treatment with dimethyl sulfoxide (DMSO), or treatment with interferon (IFN)-beta or -gamma. We found that line 1 cells expressing a transfected class I gene were significantly less metastatic than parental line 1 cells. DMSO-treated line 1 cells also formed significantly fewer metastases than parental line 1 cells. These results indicate that increased class I antigen expression decreases the metastatic potential of line 1 cells in vivo. However, we did not observe a significant decrease in the number of lung metastases in mice receiving line 1 cells treated with IFN-beta or -gamma, despite high levels of class I antigen expression. Thus, increasing class I antigen expression with IFN has an opposite effect on metastasis from class I antigen expression induced by transfection or DMSO. These results show that the method used to increase class I antigen expression is critical in terms of the in vivo effect observed. To investigate a possible mechanism for the differences observed in vivo between these class I expressing cells, we tested whether IFN alters or blocks susceptibility of line 1 cells to immune effector cells. We found IFN treatment increased the ability of line 1 cells to be recognized by CTL but concomitantly decreased the susceptibility of line 1 cells to NK cell lysis by a non-class I antigen-related mechanism. In contrast, transfected or DMSO-treated line 1 cells which were less metastatic in vivo were susceptible to both CTL and NK-mediated lysis. Taken together, these results suggest that immune intervention against metastasizing line 1 cells may involve NK cells and CTL.

Reduced tumorigenicity of a spontaneous mouse lung carcinoma following H-2 gene transfection.

Cultured cells of the murine lung carcinoma called line 1 express very low levels of H-2 class I antigens and are resistant to lysis mediated by alloreactive T cells. In order to investigate how the expression of class I antigens affects the in vivo growth of this spontaneous tumor, H-2Dp genes were transferred into line 1 cells. Cloned transfectants that displayed H-2Dp surface antigens were identified using flow cytometry. The transfected H-2Dp antigens appeared normal by two-dimensional gel electrophoresis and could also function as excellent targets for T-cell-mediated lysis in vitro. Marked differences in tumorigenicity (defined as tumor growth in immunologically competent hosts) were observed between the Dp transfected cells and untransfected or control transfected line 1 cells in syngeneic mice only if the animals had previously received injections of irradiated Dp transfectants. Expression of Dp antigens did not appreciably affect the growth of line 1 tumors in immunologically naive syngeneic mice or necessarily cause rejection in allogeneic mice. Our in vivo results show that increased expression of class I antigens can reduce the growth of tumors like line 1 that lack all class I antigens. Our results also suggest that increasing class I antigens alone on some spontaneous tumors deficient in expression will not by itself be sufficient for tumor rejection.

Properties of two forms of DNA polymerase delta from calf thymus.

Purified calf thymus DNA polymerases delta I and II each have an associated 3' to 5' exonuclease but otherwise resemble DNA polymerase alpha in size, biochemical kinetic parameters, and the presence of DNA primase [Crute, J. J., Wahl, A. F., & Bambara, R. A. (1986) Biochemistry 25, 26-36]. Here we demonstrate a functional association of polymerase and exonuclease with each delta form. Furthermore, we show that the exonuclease can be dissociated from DNA polymerase delta I but does not appear to be removable from DNA polymerase delta II. Polymerases delta I, delta II, and alpha are equally sensitive to the inhibitor aphidicolin, suggesting a similarity in active site structure. In comparison with DNA polymerase alpha and delta II, DNA polymerase delta I has intermediate sensitivity to 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) or N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP). The activity of the DNA primase of the delta II enzyme is insensitive to BuAdATP whereas 1.0 microM of this inhibitor will decrease the activity of the DNA primase of the alpha and delta I enzymes approximately 50%. Two monoclonal antibodies that potently inhibit DNA polymerase alpha are only slightly inhibitory to DNA polymerase delta I and are ineffective at inhibiting DNA polymerase delta II. DNA polymerase delta II had been previously found to be nearly inactive on nuclease-treated calf thymus DNA, relative to its activity on homopolymeric DNA. We find that addition of purified calf histone proteins or spermidine can greatly enhance synthesis by this enzyme on activated calf DNA.

Monoclonal antibodies against human ovarian tumor associated antigen NB/70K: preparation and use in a radioimmunoassay for measuring NB/70K in serum.

Murine monoclonal antibodies (MCAs) against human ovarian tumor associated antigen NB/70K have been prepared. One of these MCAs, NB12123, was chosen for the development of a radioimmunoassay for measuring serum NB/70K levels. In this assay, the average NB/70K level in 75 normal, healthy controls was 11.9 activity units (AU) with an SD of 14.9 AU. The normal cut off value for this assay was set at 45 AU (mean +2 SD). 24 of 46 (52%) ovarian cancer patients, 7 of 18 (39%) patients with benign ovarian cysts or tumors and 3 of 85 (4%) control samples had elevated serum NB/70K levels. Comparison of NB/70K levels measured in the NB12123 assay with levels measured in an assay using a polyclonal antiNB/70K previously developed in our laboratory [13] indicated that although both assays had approximately the same percentage of positive ovarian cancer patient samples, there appeared to be no correlation between the absolute NB/70K levels measured by the two assays. The rank of ovarian cancer patient samples was also different for the two assays. Also, almost 40% of patients with benign ovarian cysts and tumors had elevated serum NB/70K levels as measured by the NB12123 assay as compared to 0% for the polyclonal assay. Reciprocal cross-blocking experiments, absorption studies, and immune precipitate analysis indicated that both the monoclonal NB12123 assay and the polyclonal antiNB/70K assay measured the same population of NB/70K molecules. However, the polyclonal antibody recognizes epitopes in addition to that recognized by NB12123. Taken together, these results suggest that the epitope recognized by NB12123 is not as specific for malignant ovarian tumors as the epitope(s) recognized by polyclonal antiNB/70K and/or that more than the one epitope detected by the MCA is responsible for the specificity for ovarian cancer of the polyclonal NB/70K assay. In spite of this, the greater sensitivity and range of the monoclonal NB12123 assay make it possible to monitor serum NB/70K levels in ovarian cancer patients. In four patients examined, the fluctuating serum NB/70K levels appeared to correlate well with clinical status.

Immunoaffinity purification and properties of a high molecular weight calf thymus DNA alpha-polymerase.

A rapid, three-step purification of DNA alpha-polymerase from calf thymus is described. The key feature is immunoaffinity chromatography using a column of immobilized monoclonal immunoglobulin G (IgG) developed against human KB cell alpha-polymerase. This step is followed by preparative sucrose gradient sedimentation. The highly purified polymerase has a specific activity of 35 000 nmol of nucleotide incorporated per hour per milligram. Its molecular weight is 404 000. This molecular weight is higher than observed in some earlier purifications, possibly because salt concentrations are kept at nearly physiological levels. Also, the rapidity of purification in the presence of multiple protease inhibitors minimizes degradation. The purified enzyme is inhibited by aphidicolin, N-ethylmaleimide, and the specific monoclonal IgG, thereby identifying it as DNA alpha-polymerase. ATP at 4 mM concentration stimulates enzymatic activity up to 4-fold on calf thymus DNA templates. The enzyme is also capable of priming single-stranded DNA with RNA. The procedure represents a significant advance from purifying alpha-polymerase from calf by conventional means, since it avoids ion-exchange chromatography and harsh conditions. It also minimizes the time required to produce sufficient quantities of purified high molecular weight polymerase for analysis.

A freezing method for cell fusions to distribute and reduce labor and permit more thorough early evaluation of hybridomas.

A method is described whereby cell fusions can be bulk-frozen shortly after the hybridization step. Recoveries are shown to be comparable to those obtained for control hybridomas cultured without freezing. Advantages are discussed in terms of labor distribution and antibody assay and evaluation strategies. It is further shown that peritoneal feeder cell preparations can be conveniently frozen as a means of workload reduction.

Generating monoclonal antibodies to the opioid receptor.

BALB/c mice were immunized with an opioid receptor complex over the period of 1 year. Spleen cells from the mouse, whose serum inhibited opiate binding to rat neural membranes to the greatest extent, were fused with P3-X63-Ag8. 653.3 myeloma cells. By radioimmunoassay (RIA), 32 cell lines have been detected that secrete an antibody to a component of the isolated receptor complex. Antibodies from 2 of the cell lines have an effect on opiate binding to rat neural membranes. One antibody, OR-689.2.4 is an IgM cryoglobulin. This antibody partially inhibited the binding of 3H-dihydromorphine (3H-DHM), 3H-naloxone, 3H-ethylketocyclazocine (3H-EKC), and 3H-D-Ala2, D-Leu5 enkephalin (3H-DADLE) to rat neural membranes. The other antibody, OR-465.3, inhibited the binding of 3H-DHM and 3H-naloxone to rat neural membranes by a maximum of 70%. This antibody also inhibited the binding of 3H-DADLE to neural membranes but, did not affect the binding of this peptide to membranes from the neuroblastoma-glioma hybrid cell line, NG108-15. Work is ongoing to generate monoclonal antibodies specific for each subclass of opioid receptor.

Neurotypy: regional individuality in rat brain detected by immunocytochemistry with monoclonal antibodies.

Hybridomas from spleen cell fusions of six BALB/c mice immunized with hypothalamus were analyzed by immunocytochemistry for antibodies reactive with paraffin sections of fixed rat brain. In a total of 135 antibody producers, 60% were brain specific. Among these, 54% reacted with glial elements, pituitary cells, or basal lamina of intracerebral capillaries, with little variation among individual hybridomas in each of these groups. Forty-six percent of brain-specific antibodies reacted with neuronal structures, localizing on nerve fibers, neurofibrils, or perikarya. Neuron-specific hybridomas could be classified into groups that localized in anatomically defineable overall patterns. Within these patterns individual hybridomas exhibited extensive qualitative localization diversity ("neurotypy"). Conceivably, the genetic message for a common "proantigen" within an overall pattern may be slightly modified during differentiation of a neuron, thus leading to minor variability in antigenic expression. During antibody formation, similar minor changes occur in the differentiation of the genetic message for the antibody variable region. Apparently, minor changes in the antibody combining site among groups of hybridomas is reflected in the detectability of minor neurotypic changes among differentiated neuronal proantigens. If neurotypy proves to be the result of single-base substitutions or of variability in alignment of peptide-coding exons, the Scharrer concept of the fundamental significance of neurosecretion could also become applicable to neuronal specialization.