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Biomedical subjects

D O Chee

Publications and source records attributed to D O Chee.

At least 19 recordsLinked to original sources
PubMed1986

Continuous growth of human tumor cell lines in serum-free media.

Five human tumor cell lines were studied for growth factor requirements and for replication in serum-free media. Of the five tumor lines HT-29 (colon carcinoma), TWI (melanoma), A-549 (lung carcinoma), Panc-1, (carcinoma of the pancreas) and EJ, (bladder carcinoma) only HT-29 and TWI grew in the serum-free medium (SFM). In a series of additional experiments, a combination of transferrin (5 micrograms/ml), insulin (5 micrograms/ml), triiodothyronine (2 X 10(-10) M), epidermal growth factor (20 ng/ml), and selenium (5 ng/ml) was added to Chee's essential medium (CEM) without serum (C-TITES medium). The C-TITES modification of CEM was found to allow optimal replication of HT-29 and TWI cells. Both HT-29 and TWI cells have replicated continuously in C-TITES medium for periods of more than 15 mo. These cells replicate with slightly lower doubling times than in CEM supplemented with 10% fetal bovine serum. Deletion of insulin or transferrin from the C-TITES medium resulted in cessation of cell growth of HT-29 and TWI. HT-29 assumed a somewhat rounded morphology, whereas TWI grew with the characteristic fibroblastic morphology in C-TITES medium. Cell line EJ did not grow in C-TITES medium. The other two cell lines, A-549 and Panc-1, grew in C-TITES medium but their growth rate was much slower than that in SSM. Availability of cell lines that can be propagated in serum-free, hormone-supplemented medium may aid in the study of the mechanisms by which hormones influence cell growth.

Adenocarcinoma↗
PubMed1986

Increased detection of prolylaminopeptidase in Neisseria meningitidis by Identicult-Neisseria.

Identicult-Neisseria (Scott Laboratories, Inc., Fiskeville, R.I.), a rapid enzymatic method with chromogenic substrates, was tested in our laboratories for the identification of Neisseria gonorrhoea, Neisseria meningitidis, and Neisseria lactamica. The test correlated very highly in its identification of pathogenic Neisseria spp. with modified New York City fermentation medium. Identicult-Neisseria appeared to be more sensitive in its detection of prolylaminopeptidase activity in N. meningitidis than most of the currently available systems.

Aminopeptidases↗
PubMed1985

Cell-cycle-dependent expression of human melanoma membrane antigen analyzed by flow cytometry.

Knowledge of tumor antigenic expression is crucial to the design of therapeutic strategy. A murine monoclonal antibody (BE4) against a human melanoma membrane antigen, was used to study the in vitro expression of this antigen. By membrane immunofluorescence, BE4 reacted against 5 of 8 melanoma lines as compared to zero of 13 other cell populations. Using flow cytometry, the antigenic M14 CEM melanoma cells consisted of 40% to 60% of the total cell population. Dual-parameter measurements of DNA content and membrane antigen demonstrated that the nonantigenic cells were predominantly in G0/G1 phase, whereas the antigenic cells were distributed throughout the cell cycle. Within one passage, the sorted and recultured nonantigenic population demonstrated a similar proportion of antigenic cells as the unsorted original population. It was concluded that the expression of human melanoma antigen was cell-cycle-dependent. Understanding factors that turn off the expression of antigen in G0/G1 phase may lead to better immunotherapeutic strategies.

Antibodies, Monoclonal↗
PubMed1984

Antigenic expression of human melanoma cells in serum-free medium.

A human melanoma cell line, M14 , adapted to grow in serum free synthetic media was examined for its expression and secretion of several serologically defined melanoma associated antigens (MAA) previously described in this laboratory. Melanoma associated antigen expression and secretion was identical to that of M14 cells grown in parallel in serum supplemented medium. Spent synthetic media was found to be an enriched serum free source for the initial isolation of 100 kilodalton secreted glycoprotein MAA. M14 melanoma cells grown in synthetic media were also shown to be adaptable to the double agar clonogenic assay facilitating the examination of clonal heterogeneity in functional studies of MAA in melanoma tumor biology. Recent investigations from this laboratory have focused on characterizing human melanoma associated antigens (MAA) found either as secreted or cell surface associated glycoproteins in human melanoma cell lines. In these studies, monoclonal and polyclonal antiserums to melanoma cell components have been developed to specifically identify these MAAs immunochemically and provide a means to study the structural biochemistry of these determinants. At this time we have identified two antigens on which our research efforts are targeted: 1) a 100,000 dalton secreted glycoprotein (100K) common to melanoma, sarcoma and neuroblastoma tumor cell lines, and 2) a 250,000 dalton-high molecular weight component glycoprotein-proteoglycan complex which is thus far restricted to melanoma cells. The ultimate goal of our efforts is two-fold. Initially, we hope to develop schemes to isolate these melanoma associated antigens in sufficient quantities to obtain detailed structural information on these molecules, and secondly, we wish to implicate these glycoproteins in functional aspects of the biology of metastatic human melanoma in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, Neoplasm↗
PubMed1983

Humoral response of melanoma patients to two different tumor-associated antigens.

Sera of 8 stage II melanoma patients undergoing surgical adjunctive immunotherapy with bacille Calmette Guérin (BCG) plus melanoma cell vaccine (MCV) were assayed for humoral response by the complement-dependent antibody cytotoxicity (CDAC) assay and by the microcomplement fixation (MCF) test. The patients developed high levels of cytotoxic (CTX) and complement-fixing (CF) antibodies to the UCLA-SO-M14 (M14) cells, one of the three melanoma cell lines in the MCV. Significant rises in CTX and CF antibodies occurred one month post-immunotherapy. While the level of CTX antibodies was maintained for 11 months thereafter, the titer of CF antibodies was sustained for seven months, then gradually declined. When the sera were absorbed with lymphoblastoid (ML14) cells which are autologous to the M14 cells, two residual peaks of CTX antibodies, one and four months postimmunotherapy and two peaks of CF antibodies, one and seven months postimmunotherapy, emerged. Two sera that exhibited high levels of CTX and CF antibodies one month postimmunotherapy were absorbed with ML14 cells and human fetal brain tissue. The reactivity of one serum in both the CDAC and MCF assays were abolished, whereas the reactivity of the other serum was not significantly diminished in either assay. These data indicate that the stage II post-surgical melanoma patients developed a humoral immune response to at least two distinct tumor antigens on the membrane of the M14 cells. One of these antigens appeared to be of fetal origin (OFA), the other M14-associated (TAA). Both antigens, OFA and TAA, were involved in complement-dependent antibody cytotoxic and complement fixation reactions in vitro.

Animals↗
PubMed1982

Immunochemical and biosynthetic analysis of monoclonal antibody-defined melanoma-associated antigen.

Immunoprecipitation studies with application of monoclonal antibody F11 originally made to a partially purified spent medium antigen of melanoma cells, made it possible to delineate the molecular profiles of both the cell associated and spent medium antigens recognized on melanoma cells intrinsically labeled with glycoprotein precursors. F11 distinguishes a glycoprotein of Mr 100,000 (100 K) in the spent media of melanoma cells while a parallel analysis of detergent lysates of cells reveals a pattern of three glycoproteins of Mr 75, 77, and 100 K. Pulse-chase analysis of the biosynthesis of these antigens indicated that F11 first recognizes the 75 and 77 K antigens in the absence of a 100K component suggesting strongly that these molecules contain an antigenic site recognized by F11. The 100 K antigen appears later in the pulse-chase analysis with kinetics that suggest some of the 75 and 77 K antigens are biosynthetic precursors of the 100 K antigen. This molecule is ultimately secreted into the extracellular media and appears to be a sialoglycoprotein judging from its sensitivity to neuraminidase. A cross-reactive species with an approximate Mr 90 K is also recognized by F11 in indirect immunoprecipitation analysis of spent media from a neuroblastoma cell line indicating that a common antigenic site exists on this secreted but structurally different neuroblastoma antigen. Thus, a combination of immunochemical and biosynthetic analyses of cell-associated and secreted antigens recognized by monoclonal antibody F11 demonstrate such molecules can differ structurally when isolated from the same or different tumor cells. These findings indicate the necessity to establish molecular profiles of melanoma-associated glycoprotein antigens recognized by monoclonal antibodies to characterize and define their potential biological functions within tumor cells.

Antibodies, Monoclonal↗
PubMed1982

Mouse monoclonal antibody to a melanoma-carcinoma-associated antigen synthesized by a human melanoma cell line propagated in serum-free medium.

A monoclonal antibody, F11, was produced against a tumor-associated antigen from the spent medium of the M14 human malignant melanoma cell line which was grown continuously in serum-free medium. Ouchterlony double-diffusion study revealed that the F11 monoclonal antibody is an immunoglobulin G1. The F11 monoclonal antibody reacted positively with seven of eight (88%) melanoma, five of five (100%) carcinoma, zero to five normal, and zero of two lymphoblastoid cell lines by indirect immunofluorescence test. Also, by indirect immunofluorescence test, F11 monoclonal antibody reacted with cryostat sections from four of five (80%) melanomas, six of seven (86%) carcinomas, zero of one benign nevus, and zero of two benign breast diseases. By the indirect avidin:biotin:peroxidase complex immunoperoxidase method, the F11 monoclonal antibody reacted positively with cryostat sections from five of five (100%) melanomas, five of five (100%) breast cancers, two of two (100%) colon cancers, zero of one benign nevus, and zero of one Hodgkin's disease spleen. Thus, the tumor-associated antigen that the F11 monoclonal antibody recognizes appears to be expressed by melanomas and carcinomas, hence the designation melanoma-carcinoma-associated antigen. Microscopic observations disclosed that the melanoma-carcinoma-associated antigen is present in the cytoplasm, on the membrane of melanoma and carcinoma cells, and in the lumen of glandular structures of breast and colon carcinomas. The molecular weight of the melanoma-carcinoma-associated antigen in spent medium from the M14 CEM cell line is 100,000 as determined by sodium dodecyl sulfate:polyacrylamide gel electrophoretic analysis of indirect immunoprecipitates obtained with the F11 monoclonal antibody.

Animals↗
PubMed1980

Presence of a carcinoma-associated antigen(s) in the spent chemically defined medium of a human colon carcinoma cell line.

The human colon carcinoma cell line HT-29 was adapted to grow in chemically defined medium (CDM). The spent CDM (S-CDM) was concentrated by Amicon filtration and the crude HT-29 S-CDM purified by 40% saturated (NH4)2 SO4 precipitation. The purified antigen was tested by a microcomplement fixation (MCF) assay against the sera of cancer patients of various histologic types and against the sera of normal donors. Fifteen of 20 (75%) colon cancer, 16/20 (80%) breast cancer sera, 14/19 (74%) lung cancer sera, and 13/20 (65%) miscellaneous carcinoma sera were positive in the MCF. By contrast, 2/21 (10%) melanoma sera, 7/20 (35%) sarcoma sera, and 2/19 (11%) normal sera were positive. These data suggest the presence of a carcinoma-associated antigen in the spent CDM of the HT-29 colon carcinoma cell line adapted to grow in CDM.

Antibodies, Neoplasm↗
PubMed1978

Selective reduction of human tumor cell populations by human granulocytes in vitro.

Human peripheral blood granulocytes at a 10:1 effector cell:target cell ratio were shown by an in vitro semiquantitative staining procedure to reduce the number of human tumor cells, but not that of normal cells. Microscopic observations revealed that this selective reduction of tumor cells by granulocytes was a function of both detachment and cytolysis. The cytotoxic effect of granulocytes on the tumor cells was confirmed by a quantitative 5-[125I]iodo-2-deoxyuridine release assay. The data indicate that human granulocytes at a relatively low effector:target cell ratio (10:1) have the capacity to recognize and destroy human tumor cells in vitro.

Animals↗
PubMed1976

Detection of human tumor-associated antigens by the leukocyte migration in agarose assay.

Twenty-three of 36 (64%) lung cancer patients, 19 of 36 (54%) melanoma patients and 18 of 27 (66%) sarcoma patients tested in the leukocyte migration in agarose assay against soluble extracts of histologically similar tumors showed significant inhibition of leukocyte migration. Reactivity to extracts of dissimilar tumors was low. Sera of only 1/13 (7%) lung cancer patients, 2/19 (10%) melanoma patients and 7/21 (33%) sarcoma patients were inhibited by extracts of histologically dissimilar tumors. Only 7-9% of cancer patients reacted to paired extracts of normal tissue from the tumor donors. An average of 13% of sera from normal controls reacted to tumor extracts. Stage of disease and mode of therapy appeared to have little effect on overall reactivity in this assay, although the number of patients within the various categories was small for purposes of statistical analysis. The leukocyte migration in agarose assay shows a sensitivity and specificity to tumor-associated antigens comparable to that of the older capillary tube method in general use and may facilitate performance of migration inhibition. This assay may not be useful as a prognostic test due to the lack ofcorrelation with stage of disease and treatment modality. However, its high specificity and economical use of tumor antigen suggest applications in tumor antigen purification. The use of soluble tumor antigen preparations may make it possible to purify these antigens further to increase specificity and reactivity.

Antigens, Neoplasm↗
PubMed1976

Detection of tumor-associated antigen in human melanoma cell line supernatants.

Spent tissue culture medium (CDM-S) removed from a single cell line of human malignant melanoma grown in serum-free CDM, contained tumor-associated antigenic activity. Antibodies to CDM-S measured by complement fixation were detected in 44% (31/70) melanoma, 55% (15/27) sarcoma, 63% (24/38) carcinoma and 15% (11/72) normal sera. Delayed cutaneous hypersensitivity reactions (DCHR) were demonstrated in 4/5 melanoma patients at a 500 mug dose, 3/5 at a 100 mug dose and in 1/7 carcinoma patients at the 500 mug dose. One ml of CDM-S was shown to contain antigen equivalent to that obtained from the membranes of 2.9 X 10(7) tissue-cultured melanoma cells. After purification, 84% (16/19) sera from melanoma patients, 66% (12/18) from sarcoma and carcinoma patients and 8% (2/26) from normal controls were positive to the antigen by complement fixation.

Antibodies, Neoplasm↗
PubMed1976

Production of melanoma-associated antigen(s) by a defined malignant melanoma cell strain grown in chemically defined medium.

A human malignant melanoma cell strain, UCLA-SO-M14 (M14), was adapted to grow in serum-free, chemically defined medium (CDM). The 3 M KCl extract prepared from the CDM-grown cells (M-14-CDM) was assayed against leukocytes from melanoma patients, patients with other cancers, and normal donors by leukocyte migration inhibition (LMI). The leukocytes from 15 to 27 (56%) melanoma patients tested were LMI positive. In contrast, 4 of 18 (22%) other cancer patients and 5 of 30 (17%) normal donors leukocytes were LMI positive. One of 14 melanoma patients' leukocytes were LMI positive for a control 3 M KCI extract from autologous muscle. Comparative studies were performed with the M14-CDM extract and a 3 M KCI extract from a freshly biopsied tumor specimen from the donor of the M14 cell strain. Seven of 12 (58%) melanoma patients' leukocytes were LMI positive to the M14-CDM extract, but only 2 of 12 (17%) were LMI positive to the autologous melanoma tissue extract. Furthermore, only 100 to 300 mug protein of M14-CDM extract were required to educe delayed cutaneous hypersensitivity response in 6 of 8 (75%) melanoma patients and 0 of 5 lung cancer patients, but 500 mug protein from biopsied autologous melanoma tissue extract were needed to produce delayed cutaneous hypersensitivity response in 24 of 42 (57%) melanoma patients and 7 of 28 (25%) nonmelanoma cancer patients. These data suggest: (a) the M14-CDM cells synthesized melanoma-associated antigen(s) (MAA) in CDM; (B) the 3 M KCI extraction procedure effectively removed the MAA from the M14-CDM cells; (c) the M14-CDM cells were a more potent source of MAA than the surgical autologous melanoma specimen; and (d) the M14-CDM cells provided a continuous source of standard MAA.

Antigens, Neoplasm↗
PubMed1975

Inhibition of leukocyte migration in agarose by KCl extracts of human melanoma cell line grown in serum-free medium.

KCl extracts of Melanoma 14, a human melanoma cell line grown in chemically defined serum-free medium, inhibited leukocyte migration in 19/36 (53%) patients with malignant melanoma. Only 4/23 (17%) controls with non-melanoma malignancies and 4/25 (14%) normal subjects with no history of cancer were similarly inhibited. Only 2/27 melanoma patients tested against KCl extracts of normal muscle tissue excised from the donor of Melanoma 14 were significantly inhibited. Patients with Stage I (localized) melanoma and patients with Stage III (generalized) melanoma reacted with roughly equal frequency but the number of patients in each group was too small for meaningful statistical analysis. Leukocytes from the donor of Melanoma 14 were tested in a completely autologous system against extracts of Melanoma 14 tissue culture cells and extracts of autologous muscle and were specifically inhibited by the Melanoma 14 tissue culture extract (Migration Index = 0.67) but not by the extract of normal muscle (Migration Index = 0.96). Only 7/32 (22%) melanoma patients were significantly inhibited by an extract of non-melanoma tumor. These results suggest that melanoma-associated antigens are present in soluble extracts of this tumor line. Such extracts could provide a continuing source of standard melanoma-associated antigens for purification and chemical characterization and for diagnostic and prognostic tests in patients with malignant melanoma.

Antigens, Neoplasm↗
PubMed1975

Clinical and immunological significance of human melanoma cytotoxic antibody.

The activity of a complement-dependent cytotoxic antibody in the sera of 21 melanoma patients was investigated using a microcytotoxicity assay. Heat-inactivated sera were caused to react against mechanically dispersed fresh tumor cells in the presence of exogenous blood group AB complement. Cytotoxicity was evaluated relative to pooled normal sera as a control. Sera were cytotoxic against autochthonous tumor cells in 9 of 10 patients with localized or regional melanoma and in 1 of 11 patients with disseminated metastases. Cytotoxicity of sera was unrelated to size of tumor burden. Six of 7 antibody-positive sera (autochthonous system) were noncytotoxic to between 2 and 7 different allogeneic melanoma tumor cell preparations. Immunological reactivity of the cytotoxic antibody-positive and -negative groups was similar with respect to their capacity to be sensitized to dinitrochlorobenzene, produce positive skin tests to microbial antigens, and produce antibodies to typhoid vaccination; serum immunoglobulins were comparable. These results support the reported findings of the presence of cytotoxic antibody in the sera of melanoma patients without disseminated metastases.

Antibodies, Neoplasm↗