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At least two regions of the mouse kappa immunoglobulin gene are necessary for appropriate transcription. One is located within the J-C intron; another is located 5' of the transcription start site in all variable region gene segments and contains a highly conserved octanucleotide sequence, ATTTGCAT. Trans-acting nuclear factors have been thought to interact with these regions on the basis of studies with transient transfection of modified immunoglobulin genes into lymphoid and fibroblast cell lines. Using a sensitive gel electrophoresis DNA binding assay, we have found nuclear factors that bind to these two regulatory regions of the kappa gene. Two patterns of specific binding of nuclear factors to DNA sequences containing the J-C intron enhancer were observed. One pattern was seen in cell lines which do not express immunoglobulin, while a different pattern was observed in cell lines which actively transcribe light chain genes. We also find factors which bind to the octanucleotide containing 5' sequence which are distinct from those which bind the J-C enhancer region.

Anguidine, a protein synthesis inhibitor, has been shown previously to induce a reversible arrest of cell progression through all phases of the mitotic cycle without inducing appreciable cell kill. This "frozen" cell cycle state provided protection of Chinese hamster ovary cells against the lethal effects of 1-beta-D-arabinofuranosylcytosine, Adriamycin, hydroxyurea, 5-fluorouracil, and hyperthermia. We now report on the preferential induction of cytostasis by anguidine in normal WI-38 fibroblasts, occurring at one-tenth of the dosage required to inhibit the cycle progression of WI-38 VA13 cells, the SV40 transformant. Pretreatment with anguidine at a concentration producing effective inhibition of normal cell cycle traverse while permitting sustained proliferation of transformed cells resulted in almost complete protection of WI-38 normal cells against the growth-inhibitory effects of 1-beta-D-arabinofuranosylcytosine and Adriamycin, without reducing the antiproliferative effects of these two agents against WI-38 VA13 transformed cells. Thus, this cytokinetic concept of preferential normal tissue protection should be explored in vivo to increase the therapeutic index of cancer chemotherapy.

Cisplatin resistance remains poorly understood compared to other forms of anti-neoplastic drug resistance. In this report radiolabeled cisplatin and rapid separation techniques were used to compare drug uptake by L1210 leukemia cells that are sensitive (K25) or resistant (ZCR9) to cisplatin. Uptake of cisplatin by both cell lines was linear without saturation kinetics up to 100 microM. The resistant ZCR9 cells had 36-60% reduced drug uptake as compared to its sensitive parent line, K25. In contrast, there was no difference in the rate of efflux. We conclude that a decreased rate of uptake is one possible mechanism of cellular cisplatin resistance.

The large intron of the kappa immunoglobulin gene contains a cis-acting enhancer element, which is important in the tissue-specific expression of the gene. We have confirmed the binding activity of a sequence-specific factor present in lymphoid extracts derived from cell lines expressing, or induced to express, the kappa gene. We have extended these studies to show the binding activity is present in normal activated splenic B cells as well as lambda producing cells, and have demonstrated by DNAse footprint analysis full protection of a sequence containing the 11 bp homology to the SV-40 core enhancer. We have compared these in vitro binding studies with an analysis of protein-DNA interactions in intact murine cell lines using genomic sequencing techniques. We demonstrate significant alterations in DMS reactivity of DNA in the murine 70Z/3 cell line after it is induced to kappa expression. These alterations occur at guanine residues which are part of the the 11 bp core sequence, and are identical to those observed in cells constitutively expressing kappa. This provides direct evidence for the induced binding of the tissue specific factor to intact chromatin. In intact chromatin we also observed significant alteration in the reactivity of a guanine, 3' of the core sequence, which is part of a potential secondary DNA structure, and protection of four residues that are part of a region homologous to the heavy chain enhancer.

The human promyelocytic leukemia cell line HL-60 has an amplified number of copies of the protooncogene c-myc. It is induced to differentiate by exposure to the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). We have developed a mutant phorbol ester-tolerant (PET) line of HL-60 which undergoes a transient growth arrest but does not differentiate when exposed to TPA (Macfarlane et al., Br. J. Haematol., 68: 291-302, 1988). The defect is not due to a general failure of TPA-induced phosphorylation. In this paper, we show that exposing phorbol ester-sensitive (S) HL-60 cells to TPA caused the disappearance of the c-myc protein antigen (detected on Western blots) in 4 h, whereas TPA had no effect on the c-myc protein content of PET cells. Dimethyl sulfoxide caused the rapid disappearance of the myc antigen in both cells. PET cells had slightly more copies of the c-myc gene detected on Southern blots than S cells. c-myc mRNA was equally unstable in both cells, as determined by Northern blots following actinomycin D. TPA induced the down-regulation of c-myc mRNA in S cells to a greater extent than in PET cells. Dimethyl sulfoxide caused a rapid down-regulation of c-myc mRNA in both cell lines. This shows that PET cells have a defect in the mechanism by which protein kinase C regulates c-myc transcription. Our results provide further evidence that reduction in c-myc expression is necessary for differentiation to occur in HL-60 cells.

Flow cytometry, a technique which allows rapid quantitation of the percentages of a cell population in the various cell cycle phases, closely duplicated the previously reported circadian rhythm for mouse bone marrow DNA synthesis. The effect of ara-C, an S phase specific anti-neoplastic drug, on this rhythm was investigated. Ara-C was shown to suppress this rhythm to a much greater degree when injected at the peak of the bone marrow DNA synthesis as compared to the low. Thus, damage to the bone marrow during treatment with ara-C may be reduced if the circadian rhythm of bone marrow DNA synthesis is taken into account.

Anguidine, a protein synthesis inhibitor, has been shown to induce a reversible cell cycle arrest in exponentially growing Chinese hamster ovary cells. The effect of pretreatment with anguidine on the cytotoxicity of subsequently administered various chemotherapeutic agents, hyperthermia, and radiation was investigated. We found that anguidine greatly potentiated the cytotoxic activity of cis-dichlorodiammineplatinum(II) and melphalan by abolishing the initial shoulder and steepening the subsequent exponential portion of the survival curves. Bleomycin-induced cell kill was also potentiated by anguidine pretreatment but to a lesser extent. However, anguidine pretreatment did not substantially alter radiation cytotoxicity. In contrast, anguidine markedly reduced the lethal effect of hydroxyurea, 5-fluorouracil, and hyperthermia, three modalities with S-phase activity. To investigate whether both anguidine-induced potentiation and protection of cells by different antitumor agents were due to its induction of complete suspension of cycle traverse, experiments were also conducted with plateau-phase cultures. Whereas anguidine potentiated cis-dichlorodiammineplatinum(II) cytotoxicity in an identical fashion as noted in exponentially growing cells, its protective effect against lethal damage from Adriamycin was absent. Thus, it appears that the two opposite effects of anguidine modification of cell kill by cytotoxic agents (protection and potentiation) come about by two different mechanisms, with cell cycle arrest underlying cytoprotection and the mechanism of synergistic toxicity remaining obscure.