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E M Lord

Publications and source records attributed to E M Lord.

At least 91 records · Page 5Linked to original sources
PubMed1987

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.

Animals↗
PubMed1986

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.

Adenosine Triphosphate↗
PubMed1986

Vanadium complexes of transferrin and ferritin in the rat.

Vanadium associates with serum transferrin of rats administered vanadyl(IV) sulfate or ammonium metavanadate(V) by gastric intubation. Low molecular weight species account for only 3% of the vanadium present in plasma. The element distributes between the two major isotransferrins in proportion to their concentrations. Rat apotransferrin binds both vanadium(IV) and vanadium(V), forming 2:1 metal-protein complexes in both instances. Although the two isotransferrins apparently differ in their physiological properties, they exhibit identical vanadyl(IV) (VO2+) EPR spectra, indicating identical or very similar metal binding sites for both proteins. In contrast to other transferrins, the two sites of the rat protein are spectroscopically indistinguishable and exhibit a VO2+ EPR spectrum similar to that of the C-terminal metal binding site of human serum transferrin. VO2+ EPR signals are observed with liver, spleen, and kidney tissue samples from animals maintained on a vanadium-supplemented diet. These signals arise from a specific intracellular VO2+ complex with the iron storage protein ferritin.

Animals↗
PubMed1986

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.

Animals↗
PubMed1986

Effects of radiation on host-tumor interactions using the multicellular tumor spheroid model.

Use of the multicellular tumor spheroid as a tumor model allows separate host or tumor treatment with ionizing radiation and examination of the effects on host-tumor immune interactions. Spheroids of EMT6/Ro, a BALB/c mammary tumor were implanted into the peritoneal cavity of syngeneic immunized mice, recovered, and dissociated into single cells. Cytolytic activity of mature spheroid associated cells and peritoneal cells was resistant to radiation doses as high as 1000 rads when irradiate directly prior to assay. Mice irradiated (200, 400, 700 rads) 24 h prior to spheroid injection had an increased number of tumor cells and decreased number of tumor infiltrating and peritoneal host cells upon spheroid recovery. This was paralleled by an increased colony forming efficiency per spheroid. Cytolytic activity of the spheroid associated cells against radiolabeled EMT6 cells was in many cases decreased with radiation although lysis was the same on a per cell basis. Cytolytic activity by peritoneal cells from these mice increased with dose as measured on a per cell basis. This activity from irradiated animals was carried out by a Thy1+ cell.

Animals↗
PubMed1985

Dimethyl sulfoxide induces expression of H-2 antigens on mouse lung carcinoma cells.

The addition of dimethyl sulfoxide (DMSO) to cultures of line 1 carcinoma cells can increase the surface expression of H-2K and H-2D antigens at least 100-fold from barely detectable initial levels, as determined by using specific monoclonal antibodies and flow cytometry. H-2 values stabilize approximately 1 wk after exposure to maximally inducing concentrations of DMSO (3% vol) at densities found on normal spleen cells. Increased expression of H-2 antigens is not the result of cell selection, it is specific in that expression of an unrelated surface protein decreases, and it is associated with increased synthesis of these antigens as measured by incorporation of [35S]methionine. Additional DMSO-induced changes in the growth, cycling, lectin binding, and antigenic properties of line 1 cells are consistent with increased cell maturation. All changes are reversed when DMSO is removed. This system may facilitate study of products associated with differentiation that influence tumor cell malignancy.

Adenocarcinoma, Bronchiolo-Alveolar↗
PubMed1985

Enhanced immune recognition of H-2 antigen-deficient murine lung carcinoma cells following treatment with dimethyl sulfoxide.

Dimethyl sulfoxide (DMSO) has previously been shown to increase the surface expression of H-2K and H-2D antigens on cultured line 1 carcinoma cells. H-2 densities increase from initial levels barely detectable with flow cytometry to those found on normal BALB/c spleen cells. Here we compare the susceptibilities of untreated and DMSO-treated line 1 cells to lysis mediated by H-2d specific monoclonal antibodies and complement, cytotoxic T-cells, and natural killer cells. Induced H-2 antigens appear to function normally in that DMSO-treated cells are highly susceptible to all types of H-2 restricted immune lysis, whereas untreated line 1 cells are not. DMSO does not increase lysis of line 1 cells mediated by natural killer cells. Our results suggest that DMSO could be used to make the growth of major histocompatibility complex antigen-deficient tumors more sensitive to T-cell-mediated immunological resistance.

Animals↗
PubMed1984

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.

Animals↗
PubMed1984

Methods for using centrifugal elutriation to separate malignant and lymphoid cell populations.

Malignant cells and non-neoplastic host cells were isolated from in vivo growing solid tumors of 2 BALB/c transplantable tumors: EMT6/Ro, a mammary sarcoma, and Line 1, an alveolar cell carcinoma. The technique of centrifugal elutriation was used to separate these 2 major types of cells present within solid tumors. In addition it was found that 2 successive passages through the elutriator of the cells from the tumors resulted in a better separation of the host cells into lymphocyte and macrophage populations than was possible by an optimally designed single passage. Thus this technique can provide sufficient host cells, essentially devoid of malignant cells, for further functional analysis.

Adenocarcinoma, Bronchiolo-Alveolar↗
PubMed1984

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.

Animals↗
PubMed1984

Assessment of in situ host immunity to syngeneic tumors utilizing the multicellular spheroid model.

In situ host immunity to the EMT6/Ro mammary sarcoma tumor was evaluated by implanting multicellular spheroids of this tumor into the peritoneal cavity of syngeneic BALB/cKa mice and determining the kinetics of host cell infiltration and tumor cell killing. Spheroids grown in vitro and implanted into unsensitized mice continued to grow resulting in peritoneal tumor masses and eventual death of the animal. However, in mice previously sensitized with a single injection of heavily irradiated EMT6/Ro cells, spheroids implanted intraperitoneally were rapidly infiltrated by host immune cells (macrophages, lymphocytes, and granulocytes), tumor cell killing was detectable within 1 day and by Day 6 essentially no clonogenic tumor cells were recoverable. Despite this marked loss of both total and clonogenic tumor cells, there was little decrease in the diameter of the spheroids recovered during this time period. Physical size thus does not provide a reliable estimation of tumor cell killing. The tumor cell killing was immunologically specific in that little killing was observed when EMT6/Ro spheroids were implanted into mice sensitized with other allogeneic or syngeneic tumor cells. Host cells from within the spheroids were found to be cytotoxic for EMT6/Ro tumor cells in a 51Cr release assay. A major portion of these cytotoxic cells appear to be T lymphocytes. However, other host cell types may also be involved in the in vivo tumor cell killing.

Animals↗
PubMed1984

Heterogeneity and clonal variation related to cell surface expression of a mouse lung tumor-associated antigen quantified using flow cytometry.

Previous reports have established that line 1, a spontaneous BALB/c lung carcinoma, expresses a Mr 180,000 tumor-associated surface antigen (TSP-180). In this study, using a monoclonal antibody and flow cytometry to quantify cell surface TSP-180 expression, we found that essentially all cells in a tissue culture-adapted line 1 population express TSP-180, but that the amount of TSP-180 expressed by cells is quite heterogeneous. Variation in amount of TSP-180 was found to be in part related to cell size heterogeneity, and to the expression of TSP-180 being cell cycle-dependent. The amount of surface-expressed TSP-180 correlated somewhat with cell size, and was greater on the average for cells in the G2 cell cycle compartment. However, cells of a defined size and specific cell cycle stage still showed marked heterogeneity of expression. Even though the average amount of TSP-180 expressed per cell decreased during in vitro propagation, little change in heterogeneity was observed. To explore whether any TSP-180-related heterogeneity resulted from heritable variation of expression, 263 limiting dilution-derived line 1 clones were analyzed. The majority displayed, shortly after cloning, heterogeneous TSP-180 profiles and mean TSP-180 levels similar to those observed for the parent tumor. Occasionally, however, clones were isolated that again appeared as heterogeneous as the parent, but differed by as much as 3-fold in mean TSP-180 expression. Extensive passage did not substantially increase the low probability of isolating clones which differed in expression of TSP-180. Differences in TSP-180 expression among clones were found to be relatively stable upon passage, typically maintained after recloning, and large enough to influence clonal susceptibility to TSP-180-directed antibody and complement-mediated lysis. Heritable variation in TSP-180 expression among some clones was also shown to be independent of differences related to cell size, cell cycle, or expression of another line 1 surface antigen (P-100). We concluded that although clones demonstrating large heritable differences in TSP-180 expression can occasionally be isolated, line 1 TSP-180 heterogeneity is predominantly nonheritable, being similar to that present in recently cloned lines, quite stable during in vitro passage, and not totally accounted for by cell cycle or cell size variation.

Animals↗
PubMed1981

Cell subpopulations dispersed from solid tumours and separated by centrifugal elutriation.

The degree of non-neoplastic host-cell infiltration was assessed in 3 in vivo-in vitro tumour models commonly used in radiobiological studies: EMT6/Ro mammary carcinoma, 9L/Ro tumour and KHT sarcoma. While the 2 former tumour models have been shown to be moderately to highly immunogenic when grown s.c., the KHT sarcoma is apparently non-immunogenic. Using differential staining on single-cell suspensions from enzymatically dissociated solid tumours, all 3 tumour types were found to contain large proportions (30-60%) of non-neoplastic host cells. The actual host-cell component found in the cell suspensions differed both in type and percentage for the 3 tumours studied. These host and neoplastic cells in the cell suspensions prepared from the solid tumours could be readily separated by centrifugal elutriation. After separation the clonogenic potential of the neoplastic cells was assessed, and was found to be higher than the clonogenic capacity of the unseparated cell suspension by a factor directly related to the host/neoplastic cell ratio. Even after the removal of the host cells, the clonogenic capacities of the neoplastic EMT6 and 9L tumour cells were lower than that of the corresponding in vitro sublines (approximately 30 vs 75%). However, in the KHT sarcoma the removal of the host cell component raised the plating efficiency to approximately 60%, which was similar to the value for the in vitro cell subline of this tumour.

Animals↗
PubMed1981

Functional subsets of B cells defined by quantitative differences in surface I-A.

The quantitative expression of surface I-A determinants has been used to define functional B cell heterogeneity based on responsiveness to the trinitrophenyl (TNP) hapten on various classes of carrier molecules. Unprimed, anti-Thy 1 and complement-treated B10.A (I-Ak) B cells were stained with a monoclonal anti-I-Ak reagent that had been fluorescein (FL) conjugated. By using a fluorescence-activated cell sorter these cells were then sorted into 2 populations of cells staining heavily or lightly with this reagent. These populations of cells were then assayed for anti-TNP precursor frequency, in responses to 3 TNP-antigens, by limiting dilution analysis. B cells responsive to TNP-lipopolysaccharide (LPS) and TNP-sheep red blood cells (SRBC) were found in both the B cells that expressed a low amount of surface I-A and the B cells that expressed a high amount of I-A. However, B cells with the ability to respond to TNP-Ficoll were found only in the B cells that expressed a low amount of surface I-A. These experiments suggest that the quantitative expression of surface I-A can be used to define B cell populations with different abilities to respond to hapten coupled to Ficoll.

Animals↗
PubMed1980

The multicellular tumor spheroid model. II. Characterization of the primary allograft response in unsensitized mice.

The multicellular spheroid model previously has been used to characterize secondary allograft responses in sensitized mice. In this study multicellular spheroids of EMT6 mammary sarcoma were implanted into unsensitized allogeneic mice to assess sensitization, graft infiltration, and functional activities of host cells in a primary allograft response. During the first 5 days after implantation, the spheroids continued to grow similar to controls maintained in vitro. After 6 days the spheroids decreased in size, and recovery of clonogenic tumor cells fell markedly. Cytotoxic effector cells active in an in vitro 51Cr release assay were recoverable from the spheroids beginning at day 4 and reached a maximum at day 7. Lymphocytes and macrophages were present in the largest numbers at days 7 to 8. The predominant cytotoxic cell appears to be a T lymphocyte.

Animals↗