Bleomycin and X-ray-hypersensitive Chinese hamster ovary cell mutants: genetic analysis and cross-resistance to neocarzinostatin

We have previously reported the isolation of 3 mutants of Chinese hamster ovary cells which exhibit hypersensitivity to bleomycin. 2 mutants were isolated on the basis of bleomycin-sensitivity [designated BLM-1 and BLM-2, Robson et al., Cancer Res., 45 (1985) 5304-5309] and 1 as adriamycin-sensitive [ADR-1, Robson et al., Cancer Res., 47 (1987) 1560-1565]. Because bleomycin generates DNA-strand breaks via a free-radical mechanism, we have studied the survival response of these mutants to a range of drugs which also generate free radicals and consequently DNA-strand breaks. The mutants are all hypersensitive to phleomycin, which differs from bleomycin in being unable to intercalate due to a modified bithiazole moiety. However, BLM-2 cells alone are hypersensitive to pepleomycin, a semi-synthetic bleomycin analogue. In contrast, BLM-1 cells are more sensitive than BLM-2 to streptonigrin (which operates via a hydroquinone intermediate). ADR-1 cells show wild-type resistance to streptonigrin. The results obtained with neocarzinostatin, an antibiotic requiring thiol activation, are unusual in that both BLM-1 and BLM-2 are approximately 3-fold more resistant than parental cells. However, the steady-state intracellular level of the major non-protein thiol, glutathione, is not altered in BLM-1 or BLM-2 cells. ADR-1 cells show essentially wild-type resistance to neocarzinostatin. Analysis of cell hybrids shows that BLM-1 and BLM-2 cells are phenotypically recessive in combination with parental CHO-K1 cells and represent different genetic complementation groups not only from one another, but also from the bleomycin-sensitive mutant xrs-6, isolated on the basis of X-ray sensitivity by Jeggo and Kemp [Mutation Res., 112 (1983) 313-319]. These results indicate that at least 3 gene products are involved in cellular protection against bleomycin toxicity in mammalian cells.     

Primary structure of a Plasmodium falciparum malaria antigen located at the merozoite surface and within the parasitophorous vacuole

DNA encoding an antigen of 101,000 apparent molecular weight from the human malaria parasite Plasmodium falciparum was cloned and sequenced. Genomic DNA from the Camp strain covering the complete coding region along with cDNA from the FCR3 strain covering 81% of the coding region were obtained. The cloned DNA specified a full-length protein of 743 amino acids which included two tandemly repeated regions, one near the amino terminus containing eight hexapeptide repeats of sequence TVNDEDED, and the second near the carboxyl terminus containing primarily KE and KEE repeats. The latter repeated region is encoded by a 174-base stretch of mRNA containing only a single pyrimidine. Except for a putative leader sequence located at the amino terminus of the protein, the protein is hydrophilic and highly charged with a calculated isoelectric point of 5.6. Sequences from the Camp and FCR3 strains are very close and are also nearly identical to the partial cDNA sequence of the acidic basic repeated antigen (ABRA) protein from the FC27 strain (Stahl, H.D., Bianco, A.E., Crewther, R.F., Anders, R.F., Kyne, A.P., Coppel, R. L., Mitchell, G.F., Kemp, D.J., and Brown, G.V. (1986) Mol. Biol. Med. 3, 351-368). ABRA was previously shown to be located at the merozoite surface and in the parasitophorous vacuole. Because of its location and because it becomes complexed to merozoites when schizonts rupture in the presence of immune serum, ABRA is a candidate component of a malaria vaccine.     

Transforming growth factor-beta is a potent immunosuppressive agent that inhibits IL-1-dependent lymphocyte proliferation

Transforming growth factor-beta (TGF-beta), a product of neoplastic and hemopoietic cells, is a bifunctional regulator of the immune response. At femtomolar concentrations, TGF-beta stimulates monocyte migration, and picomolar quantities induce synthesis of monocyte growth factors, including IL-1, that may promote tissue repair by regulating fibrosis and angiogenesis. Paradoxically, TGF-beta at picomolar concentrations also blocks the ability of IL-1 to stimulate lymphocyte proliferation. At 0.01 to 1.0 ng/ml, TGF-beta 1 and its homologue, TGF-beta 2, suppress the IL-1-dependent murine thymocyte proliferation assay. TGF-beta also inhibits human peripheral blood T lymphocyte mitogenesis. Inhibition of cell division appears to occur after activation of the lymphocytes inasmuch as neither gene expression nor translation of IL-2R is suppressed. Furthermore, TGF-beta does not block synthesis of IL-2. Therefore, TGF-beta 1 and TGF-beta 2 likely act at a site distal to IL-1 to block lymphocyte DNA synthesis. These findings suggest that TGF-beta secreted in an inflammatory site may be beneficial in diminishing lymphocyte function while promoting fibrosis and tissue repair. However, TGF-beta generated by neoplastic tissues may provide a mechanism for unrestricted tumor cell growth through its selective immunosuppressive effects.     

Complete exon-intron organization of the human leukocyte common antigen (CD45) gene

Ten genomic DNA clones encoding the human leukocyte common Ag (LCA, CD45) gene were isolated by screening human genomic DNA libraries with LCA cDNA probes. One genomic DNA clone contains the promoter region and the first two exons, as determined by primer extension analyses and S1 nuclease protection studies as well as nucleotide sequence determination. The first exon does not encode a peptide, while the second exon contains the initiation ATG codon and encodes the signal peptide. The other nine genomic DNA clones, which are separated from the first genomic clone by an unknown distance, are connected and span a total of 73 kb. The nine connected genomic clones encode a total of 31 exons. The 33 exons encoded by these 10 genomic clones account for the entire cDNA sequences including the 5' and 3' untranslated sequences. Exon 3 and exons 7 through 15 encode the extracellular domain sequences that are common to all LCA isoforms. Differential usage of exons 4, 5, and 6, generates at least five distinct LCA isoforms. Exon 16 encodes the transmembrane peptide. The cytoplasmic region of the leukocyte common antigens is composed of two homologous domains. Exons 17 through 24 encode the first domain, and exons 25 through 32 encode the second domain. The comparison of these exons indicated that the homologous domains were generated by duplication of several exons. The most 3' exon (exon 33) encodes the carboxy terminus of the LCA molecules and includes the entire 3' untranslated sequence.     

Thymosin fraction 5 (TF5) stimulates secretion of adrenocorticotropic hormone (ACTH) from cultured rat pituitaries

In vivo administration of a partially purified thymic hormone-containing extract of the thymus gland, TF5, causes an increase in serum glucocorticoids. The lack of a direct effect of TF5 on adrenal corticosterone secretion suggests that it is mediated at the level of the pituitary. Cultured rat pituitary monolayers were used to determine if the effect is mediated by stimulation of ACTH secretion from the pituitary. Two lots of TF5, BPP100 and C114080-01, caused a dose dependent secretion of ACTH from cultured pituitary monolayers. There was a synergistic effect when the cells were treated with both TF5 and corticotropin-releasing factor (CRF). Immunoneutralization studies were done in which the cells were treated with TF5 or CRF and an antibody to CRF. The antibody completely blocked CRF induced ACTH release, but had no effect on TF5 stimulated ACTH release, suggesting that the activity is not due to a CRF-like peptide in TF5. A number of peptides isolated from TF5, and certain other peptides produced by the immune system were evaluated for their ability to stimulate ACTH secretion. These included thymosin (TSN) alpha 1, alpha 11, and beta 4, prothymosin alpha (PT alpha, thymopoeitin 5 (TP5), factuer thymique serique (FTS), interferon alpha (INF alpha), INF gamma, interleukin 1 (IL-1), and interleukin 2 (IL-2). None of these factors had any effect on pituitary ACTH secretion. These results demonstrate that some peptide component of TF5 causes an increase in serum corticosteroids by stimulating pituitary ACTH release.     

Mutation induction and relative biological effectiveness of neutrons in mammalian cells. Experimental observations

The induction of mutation by graded doses of monoenergetic neutrons was examined using the human-hamster hybrid cell system. The AL cells, formed by fusion of human fibroblasts with the gly- A mutant of the Chinese hamster ovary cells, contain the standard set of hamster chromosomes plus a single human chromosome, number 11. These cells contain specific human cell surface antigens that render them sensitive to killing by specific antisera in the presence of complement. Mutant AL cells that have lost the surface markers, however, would survive and give rise to scorable colonies. The cells were irradiated with neutrons produced at the Radiological Research Accelerator Facility of Columbia University. Doses corresponding to low, moderate, and high cytotoxicities and in energies ranging from 0.33 to 14 MeV were used. Neutrons induced a dose-dependent cytotoxicity and mutation frequency in the AL cells. Over the range of doses examined, it was found that the mutagenesis induced by neutrons was energy-dependent and the frequencies were a curvilinear function of dose for both the a1 and a2 antigenic loci examined. In comparison to gamma rays, the relative biological effectiveness (RBE) for cell lethality at the 10% survival level ranged from 5.2 for 0.33 MeV to 1.8 for 14 MeV neutrons. The RBE for mutation induction at the a1 locus, however, ranged from 30 for 0.33 MeV to 4.2 for 14 MeV neutrons at or around the lowest levels of effect examined. Results of the present study demonstrated that neutrons, when measured under conditions which permit detection of a spectrum of gene and chromosomal mutations, in fact, are more efficient mutagens than previously thought.     

Oncogenic transformation by fractionated doses of neutrons

Oncogenic transformation was assayed after C3H 10T1/2 cells were irradiated with monoenergetic neutrons; cells were exposed to 0.23-, 0.35-, 0.45-, 5.9-, and 13.7-MeV neutrons given singly or in five equal fractions over 8 h. At the biologically effective neutron energy of 0.45 MeV, enhancement of transformation was evident with some small fractionated doses (below 1 Gy). When transformation was examined as a function of neutron energy at 0.5 Gy, enhancement was seen for cells exposed to three of the five energies (0.35, 0.45, and 5.9 MeV). Enhancement was greatest for cells irradiated with 5.9-MeV neutrons. Of the neutron energies examined, 5.9-MeV neutrons had the lowest dose-averaged lineal energy and linear energy transfer. This suggests that enhancement of transformation by fractionated low doses of neutrons may be radiation-quality dependent.     

Venous occlusion pressure and vascular permeability in the dog lung after air embolization

Pulmonary edema has frequently been associated with air embolization of the lung. In the present study the hemodynamic effects of air emboli (AE) were studied in the isolated mechanically ventilated canine right lower lung lobe (RLL), pump perfused at a constant blood flow. Air was infused via the pulmonary artery (n = 7) at 0.6 ml/min until pulmonary arterial pressure (Pa) rose 250%. While Pa rose from 12.4 +/- 0.6 to 44.6 +/- 2.0 (SE) cmH2O (P less than 0.05), venous occlusion pressure remained constant (7.0 +/- 0.5 to 6.8 +/- 0.6 cmH2O; P greater than 0.05). Lobar vascular resistance (RT) increased from 2.8 +/- 0.3 to 12.1 +/- 0.2 Torr.ml-1.min.10(-2) (P less than 0.05), whereas the venous occlusion technique used to determine the segmental distribution of vascular resistance indicated the increase in RT was confined to vessels upstream to the veins. Control lobes (n = 7) administered saline at a similar rate showed no significant hemodynamic changes. As an index of microvascular injury the pulmonary filtration coefficient (Kf) was obtained by sequential elevations of lobar vascular pressures. The Kf was 0.11 +/- 0.01 and 0.07 +/- 0.01 ml.min-1.Torr-1.100 g RLL-1 in AE and control lobes, respectively (P less than 0.05). Despite a higher Kf in AE lobes, total lobe weight gains did not differ and airway fluid was not seen in the AE group. Although air embolization caused an increase in upstream resistance and vascular permeability, venous occlusion pressure did not increase, and marked edema did not occur.     

Natural course of penicillamine nephropathy: a long term study of 33 patients

To elucidate the natural course of the nephropathy associated with penicillamine and thereby facilitate its clinical management 33 patients with rheumatoid arthritis who developed proteinuria during treatment with oral penicillamine were studied in detail throughout their renal illness. Renal biopsies were performed, and creatinine clearance and proteinuria were measured serially for 74 months (range 16-148 months). Fourteen patients developed proteinuria within six months after the start of treatment and 27 within 12 months. When treatment was stopped the proteinuria reached a median peak of 4·2 g/24 h (range 0·3-15·0 g/24 h) at one month (range 0-7 months) before resolving spontaneously by six months (12 patients), 12 months (21), or 18 months (29). In all patients but one, who developed carcinoma of the renal pelvis, proteinuria resolved by 21 months and its median duration was eight months. The median first and last measurements of creatinine clearance showed no appreciable change (80 ml/min and 78 ml/min), and no patient died from or needed treatment for renal failure. The HLA-B8 or HLA-DR3 alloantigen, or both, were identified in 10 patients. Renal biopsy specimens showed membranous glomerulonephritis in 29 patients, minimal change nephropathy in two, and electron dense deposits in the mesangial regions in two.In all the patients whose nephropathy was due solely to treatment with penicillamine the proteinuria resolved completely when the drug was withdrawn; renal function did not deteriorate, and corticosteroids were unnecessary.