The major histocompatibility complex class I heavy chain as a structural subunit of the human cell membrane insulin receptor: Implications for the range of biological functions of histocompatibility antigens ( ABSTRACTMonoclonal antibodies against some of the monomorphic determinants of major histocompatibility complex (MHC) class I molecules reduce insulin binding and precipitate '251-labeled insulin receptor preparations. A monoclonal antibody with specificity for the insulin binding site on the cell membrane insulin receptor of human cells was used to precipitate insulin receptors from human cell lines and resulted in distinct bands of Mr 1l30,000, 90,000, and 45,000. The Mr 45,000 molecules thus precipitated were subjected to

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The diversity in insulin receptor expression within eukaryotic cell populations can be studied with fluorochrome conjugated reagents with high affinity to the insulin receptor in combination with flow cytometry. We studied the optimal conditions for application of fluorescein isothiocyanate (FITC) conjugated insulin in combination with the fluorescence activated cell sorter (FACS) to analyse insulin receptor expression, and also studied the feasibility of this method for identifying and isolating viable subsets with differences in insulin receptor expression within a cell population. Semisynthetic human insulin was conjugated to FITC, which resulted in at least four types of FITC-insulin molecules with different affinities to the insulin receptor. Each type of FITC-insulin was isolated by semipreparative reverse phase high pressure liquid chromatography. The preparation with a fluorescein/ protein ratio of -1.0 was found to have the highest affinity to the receptor, the highest biological activity (-50% of native insulin), and similar antigenicity as native insulin. The optimal staining conditions with respect to pH, time of incubation, and cell number were determined, and were different in some aspects from labelling with 125I-insulin. The binding of FITCinsulin to cells was saturable and could be displaced with unlabelled insulin. The fluorescence signal could be converted to absolute numbers of fluorescein molecules by a calibration curve, and the absolute number of specifically bound FITCinsulin molecules calculated from a F/P ratio -1.0. The FITC-insulin/FACS method permits estimation of the total number of insulin receptors (high plus low affinity), and the data obtained correlate well with the results from Scatchard plot of 125I-insulin binding data. However, the latter method also permits selective detection of the number of high affinity insulin receptors, which cannot be done with FITC-insulin/ FACS that has a lower level for detection of fluorescence on -2000-3000 fluorescein molecules per cell. The FITC-insu-Iin/FACS methodology permitted identification and isolation of viable cellular subsets within a cell population as based on the number of insulin receptors and was also used to study variations in insulin receptor density in human leucocytes. The method should make it possible to perform a number of hitherto unfeasible analyses of the biology of insulin receptor expression on eukaryotic cells.

Liquid medium cultures of three human cell lines (B-lymphoma, myeloma, and squamous lung carcinoma) with population-doubling times (PDT) and cloning efficiencies (CE) in the range of 32-43 h and 0.01-5.6%, respectively, were exposed to 5-azacytidine (5-azaC) for 3 d. The doses used (1-3/zM) were found to be nontoxic as measured by cell growth in liquid and semisolid agar medium and to be nonmutagenic as measured by the rate of generation of ouabain-and 6-thioguanine-resistant cell variants. After 5-azaC treatment, cell samples were subsequently harvested every day and assayed for their CE in semisolid agar medium. For each cell line, 30 to 42 individual clones were harvested at the day of maximal CE and expanded in liquid culture medium. PDT and CE were determined for each subclone about every 6 wk for 12 too. The majority of the subclones had unaltered PDT and CE compared to the original lines. However, several clones had profoundly changed proliferative activity with PDT on ~12-14 h and/or CE 5 to >50%. Some of the clones with altered growth properties reverted to PDT and/or CE values of untreated clones. However, a few clones of each line had stable alterations with PDT on 12-14 h and CE 5 to >50%; these clones were all significantly hypomethylated. It is concluded that the human gene repertoire does contain genes that appropriately activated can result in growth properties with very short PDT and high CE (and comparable to animal cell lines), and that this activation may be obtained by 5-azaC treatment. It is conceivable that the procedure here described to alter growth properties of human cell lines may be applied to experimental situations, where alterations of cell growth properties are desired.Many reports in recent years on regulation of both cellular 0-6) and viral~ genes (7-11) have provided experimental support to the hypothesis (12-15) that 5-methylcytosine (mSCyt) t may be important in the regulation of gene expression. Moreover, a number of experiments have shown that exposure of cells to 5-azacytidine (5-azaC) may result in hypomethylation of DNA and cause expression of otherwise silent genes of both cellular and viral origin (for review, cf. references 16, 17) to the extent that it mimics mutation induced genetic alterations, e.g., reversion frequency of thyAbbreviations used in this paper." 5-azaC, 5-azacytidine; CE, cloning efficiency; mSCyt, 5-methylcytosine; Oua r, ouabin resistant; PDT, population-doubling time; RPMI/FCS, RPMI-1640 medium SUlSplemerited with 0.3% L-glutamine and 10-15% fetal calf serum; 6-TG ~, 6-thioguanine resistant. midine kinase negative cells to thymidine kinase positive cells (18).The growth properties of malignant tumor cell lines are at least in part reflected in the growth rate and CE. However, it has hitherto not been possible by exogenous factors to alter profoundly these biological features, despite numerous attempts that mainly have been based on alterations in the growth medium conditions such as addition of growth factors like insulin and transferr...