IgM+ cells cultured from the I.29 B cell lymphoma can be induced with lipopolysaccharide (LPS) or, to a greater extent, with LPS plus anti‐idiotype antibody to switch to IgG2a, IgE or IgA expression. The isotype switch is accompanied by rearrangement of immunoglobulin (Ig) heavy (H) chain genes. Here we demonstrate that the commitment of the I.29 IgM+ cells to switch to IgA appears to be manifested by hypomethylation of the alpha constant region genes in IgM+ cells, and by the presence of small amounts of RNAs transcribed from non‐rearranged alpha gene(s) in IgM+ cells. The commitment to switch to IgE or IgG2a is also in accord with the presence of small amounts of RNA transcripts from the non‐rearranged epsilon and gamma 2a genes, although the hypomethylation of the epsilon and gamma 2a genes is not as dramatic as that of the alpha genes. These results suggest that I.29 cells switch specifically to IgA, IgE or IgG2a due to the activation of the corresponding H chain constant region genes in IgM+ cells prior to the actual switch recombination event.

A cDNA encoding the human leukocyte antigen CD59 has been isolated from the erythroid cell line K-562 and its identity confirmed through expression in COS cells. Northern blotting reveals three message species of approximately 800, 1400 and 2000 bases in size, which are constitutively expressed in all lymphoid, erythroid, myeloid, and neural cell types tested thus far. Southern blotting of human DNA indicates a pattern consistent with the presence of a single gene, which has been mapped to chromosome 11 by somatic cell hybrids. Also, the finding of a transcriptionally active cross-hybridizing gene in monkey cells suggests conservation of CD59 sequences among primates. Comparison of the CD59 protein sequence with those of the Ly-6E and Ly-6C antigens discloses a similarity in overall structure, including the alignment of abundant cysteine residues, hydrophobic carboxy termini and conservation of amino acids surrounding the proposed phosphatidylinositol-glycan modification site for Ly-6 molecules. Unlike Ly-6, however, CD59 expression does not appear to be inducible with interferons. This, along with its limited homology and different tissue distribution, cast doubt upon the functional equivalence of CD59 and either of the well-characterized mouse Ly-6 proteins.

L1210 cell variants resistant to edatrexate (EDX) were isolated by selection in vivo during therapy with this folate analogue. Among the variants selected, seven (L1210/EDX-4 to -7 and L1210/EDX-12 to -14) were found to exhibit 2-23-fold lower levels of folylpolyglutamate synthetase (FPGS) activity compared with parental L1210 cells. Lower levels of FPGS activity in cell-free extract from these variants using EDX as substrate were characterized by the same relative decrease in value for Biochemical alterations associated with acquired resistance of tumor cells to classical folate analogues are highly diverse (1-5). This phenotypic diversity most likely reflects the common occurrence of multiple genomic alterations among clonal variants selected for resistance to these agents either in vitro or in vivo. As a consequence, acquired resistance to methotrexate and newer folate analogues under development will remain (6 -8) a major limitation to their effective clinical utility. One of the determinants of cytotoxicity shared (9 -11) by most folate analogues, whether targeted to dihydrofolate reductase or folatedependent biosynthetic enzymes, is the process of intracellular polyglutamylation mediated by the enzyme folylpolyglutamate synthetase (FPGS).1 This process is not only important to the conservation and efficient utilization by proliferating cells of folate coenzymes (12-18) but is responsible, as well, for converting folate analogues (9 -18) to longer chain polyglutamate anabolites that are more retentive and more effective inhibitors of folate-dependent enzymatic targets. Subsequently, examples have been reported from our laboratory (4, 19) and elsewhere (20, 21) of tumor cell variants with acquired resistance to methotrexate and other folate analogues that exhibit lower levels of FPGS activity when compared with parental cells and are cross-resistant to most other classical folate analogues. In one of our own studies with the L1210 leukemia, resistant variants (L1210/EDX) were independently selected (4) during therapy of tumor-bearing mice with edatrexate (EDX). Some of these variants exhibited levels of FPGS activity that were substantially lower than in parental cells. In the current studies, we address the underlying basis for these alterations in FPGS activity in these resistant variants at the level of the FPGS protein elaborated by these cells as well as cognate gene expression. Our results appear to define a novel mechanism underlying these modifications in FPGS activity in these variants that may have broad significance in regard to the regulation of FPGS gene expression and for therapy with classical folate analogues. A preliminary report of some of these findings has already been presented (22) in abstract form. EXPERIMENTAL PROCEDURESCells and Culture Conditions-Methods for the isolation of variants of the L1210 cell used in these studies were described in detail earlier (4). These variants were independently isolated from ascite fluid in the peritoneal cavity of mice following treatment with EDX. ...

The murine B cell lymphoma I.29 contains cells expressing surface IgM or IgA with identical heavy chain variable regions (9, 25, and D. Klein and J. Stavnezer, unpublished data). Purified IgM+ cells from the lymphoma have been adapted to culture and induced to switch to IgA, IgE, or IgG2 by treatment with lipopolysaccharide (LPS) or by treatment with a monoclonal anti-I.29 antiidiotype plus LPS. Clones of IgM+ cells have been obtained and induced to switch. Under optimal conditions, 30% of the cells in the culture expressed IgA 8 d after the inducers were added, and by 15 d 90% of the cells were IgA+. In actively switching cultures, up to 50% of the cells whose cytoplasm stained positively with anti-IgA stained simultaneously with anti-IgM, which indicates that the appearance of IgA+ cells in the cultures was due to isotype switching and not to clonal outgrowth. Examination by Southern blotting experiments of the Ig heavy chain genes in I.29 cells before and after switching revealed that isotype switching was accompanied by DNA recombinations that occurred within or immediately 5' to the tandemly repeated switch sequences. Within 3 d after the addition of inducers of switching, the nonexpressed chromosome underwent a variety of deletions or expansions within the S mu region, and a portion of the S alpha regions had undergone a 0.9-kb deletion. In cultures that contained at least 12% IgA+ cells, rearranged, expressed alpha genes, produced by recombination between the S mu region within the expressed mu gene and the S alpha region, were detected.

Growth of one of these variants (L1210/R69), with metoprine in the presence of decreasing concentrations of l,L5-CHO-folateH 4 (natural diastereoisomer of 5-formyltetrahydrofolate), resulted in the selection of other variants (L1210/R82, R83, and R84) with further reduction in one-carbon, reduced folate transport and in two cases (L1210/R83 and R84) with 3-8-fold increased folylpolyglutamate synthetase (FPGS) activity and folate compound polyglutamate formation in situ. Metoprine resistance was further increased, and the requirement for exogenous folate during growth was decreased as well in these variants. The increase in FPGS activity observed in L1210/R83 and R84 was characterized by 3-and 8-fold increases in value for V max with no change in K m and the same increase in a 60 -61-kDa protein as shown by immunoblotting. Northern blotting revealed the same increases in these two variants in the level of a 2.3-kilobase FPGS mRNA when compared with control, while Southern blotting of genomic DNA did not reveal any increase in FPGS gene-copy number or restriction polymorphisms. Also, no difference in stability of FPGS mRNA was found between parental and variant cells. In contrast, nuclear run-on assays revealed differences among these cell types in the rate of FPGS mRNA transcription that correlated with increased FPGS activity, protein, and mRNA level in the variants. Similar studies with a transport-defective, methotrexate-resistant L1210 cell variant (L1210/R25) documented a 2-3-fold decrease in FPGS activity, protein, and mRNA levels that was accounted for by a decrease in FPGS mRNA transcription. These results provide the first examples of constituitively altered transcriptional regulation of FPGS activity associated with acquired resistance to antifolates.Cellular folates exist primarily as ␥-polyglutamate peptides (1-5) of varying chain length. Their metabolism to polyglutamates and that of folate analogues are mediated (1-5) by the enzyme, folylpolyglutamate synthetase (FPGS), 1 and metabolic turnover of these anabolites appears to be modulated by folylpolyglutamate hydrolase after their mediated entry into lysosomes (for review, see Ref. 6). In tumors and normal proliferative tissues of animals and man, the process of polyglutamylation has pharmacologic relevance with respect to the cytotoxicity (8, 9) and therapeutic utility (7-13) of classical folate analogues. Also, both decreased levels of FPGS activity (14, 15) and increased levels of folylpolyglutamate hydrolase activity (16) have been associated with acquired resistance to these analogues. The mechanistic basis for these alterations remain to be elucidated.The process of folylpolyglutamylation in normal proliferative and neoplastic mammalian tissues is important (1-5) to the conservation and efficient utility of folate coenzymes that are required for one-carbon transfer reactions during macromolecular biosynthesis. Consequently, levels of FPGS activity appear to be highest in the proliferative fraction of normal differentiating tissues (7,(17)(18)(...