Addressing parenting concerns of bone marrow transplant patients: opening (and closing) Pandora's box.

Although a significant number of adults undergoing stem cell transplant (SCT) or bone marrow transplantation (BMT) care for dependent children, and these treatments pose significant challenges for families, research has virtually ignored the impact of parenting on patients' quality of life during BMT/SCT and children's responses to having a parent undergo these treatments. Physicians rarely inquire about parenting concerns related to the extended hospitalizations necessitated by these treatments, yet clinical experience suggests that addressing patient concerns about children's reactions to cancer and BMT/SCT can improve the experience of the patient and the patient's family, and help the medical team respond effectively to sources of patients' distress. Parents frequently want to know what reactions to expect from children, thus general developmental information is reviewed, and recommendations given for when professional help for children is warranted. A key way for parents to support their children is with open, honest communication; however, parents often find it extremely difficult to talk about cancer and BMT/SCT with their children. The medical team can assist patients' efforts to communicate with and support their children by asking about a patient's children, providing some targeted information, and discussing the potential impact of treatments on parenting capacity. Inquiring about and addressing parenting concerns may initially seem difficult, but can ultimately facilitate stronger doctor-patient alliances, and more compassionate care.

Overexpression of genes involved in vesicular trafficking to the vacuole defends against lethal effects of oxidative damage.

Anticancer bleomycins and structurally-related analogs are oxidative agents that mimic ionizing radiation in many of their cellular effects. The current study was designed to better understand this class of radiomimetic and oxidative drugs, and how cells defend against them to become resistant. Based on some of the properties conferred by the blm5-1 mutation of Saccharomyces cerevisiae, a multi-step cloning strategy was developed to search for genes that protect cells against oxidative damage and lethal effects of bleomycin treatments. The strategy employed blm5-1 mutant strains to search for genes that rescued the drug hypersensitivities conferred by the mutation, and utilized the inability of homozygous blm5-1 mutant diploid strains to grow at elevated temperatures. This approach identified the VPS3, VPS8 and PEP7 genes that function in vesicular trafficking between the endosome and the yeast vacuole via the carboxypeptidase Y (CpY) pathway. Mutant blm5-1 strains possess several phenotypic characteristics consistent with CpY mutants, including reduced mitotic growth rates and sporulative abilities. However, blm5-1 strains were not found to be defective in the transport of CpY into the vacuole. We suggest that the ability of the VPS3, VPS8 and PEP7 genes to rescue lethal effects of oxidative damage resulted from the overexpression of these genes.

Pleiotrophic cellular deficiencies conferred by the blm5-1 mutation of Saccharomyces cerevisiae.

Mutational alteration of the BLM5 gene of the model eukaryote, Saccharomyces cerevisiae, confers extreme hypersensitivities to lethal effects of ionizing radiation, anticancer bleomycins and structurally-related phleomycins. Additional properties conferred by the blm5-1 mutation in haploid and diploid strains were investigated for the current report. Only one copy of blm5-1 together with the normal BLM5 allele was sufficient to produce mitotic and meiotic defects in diploids, and greatly increase killing by bleomycin beyond wild type levels. Mitotic growth rates of blm5-1/blm5-1 homozygous mutant strains were slower than wild type or BLM5/blm5-1 heterozygous strains at 30 degrees C, and growth was nearly completely inhibited at 37 degrees C. Meiosis was inhibited at 30 degrees C and 37 degrees C in mutant homozygotes, and at 37 degrees C in BLM5/blm5-1 heterozygotes, while meiosis occurred at equivalent frequencies in wild type strains at both temperatures. Surprisingly, mutant strains were found to associate extremely low quantities of [S-methyl-3H]bleomycin A2, in contrast to normal strains that associated quite high amounts. However, the fractions of the total associated radioactivities that were released from normal and blm5-1 cells were equivalent. These results suggested that the extremely high killing suffered by blm5-1 mutant strains in response to bleomycin treatments results from something other than increased intracellular drug concentrations.

Soluble 2-substituted aminopyrido[2,3-d]pyrimidin-7-yl ureas. Structure-activity relationships against selected tyrosine kinases and exploration of in vitro and in vivo anticancer activity.

In continuing our search for medicinal agents to treat proliferative diseases, we have discovered 2-substituted aminopyrido[2,3-d]pyrimidin-7-yl ureas as a novel class of soluble, potent, broadly active tyrosine kinase (TK) inhibitors. An efficient route was developed that enabled the synthesis of a wide variety of analogues with substitution on several positions of the template. From the lead structure 1, several series of analogues were made that examined the C-6 aryl substituent, a variety of water solublizing substitutents at the C-2 position, and urea or other acyl functionality at the N-7 position. Compounds of this series were competitive with ATP and displayed submicromolar to low nanomolar potency against a panel of TKs, including receptor (platelet-derived growth factor, PDGFr; fibroblast growth factor, FGFr;) and nonreceptor (c-Src) classes. Several of the most potent compounds displayed submicromolar inhibition of PDGF-mediated receptor autophosphorylation in rat aortic vascular smooth muscle cells and low micromolar inhibition of cellular growth in five human tumor cell lines. One of the more thoroughly evaluated members, 32, with IC50 values of 0.21 microM (PDGFr), 0.049 microM (bFGFr), and 0.018 microM (c-Src), was evaluated in in vivo studies against a panel of five human tumor xenografts, with known and/or inferred dependence on the EGFr, PDGFr, and c-Src TKs. Compound 32 produced a tumor growth delay of 14 days against the Colo-205 colon xenograft model.

The novel BLM3 gene encodes a protein that protects against lethal effects of oxidative damage.

Mutational alteration of the BLM3 gene in Saccharomyces cerevisiae confers hypersensitivities to lethal effects of ionizing radiation, anticancer bleomycins and structurally-related phleomycins. Bleomycin is used clinically in the treatment of many types of cancers, including Kaposi's sarcoma. The BLM3 gene was cloned from a genomic library by complementing the drug hypersensitivities conferred by the codominant blm3-1 mutation. The nucleotide sequence of BLM3 encodes a predicted integral protein of 1804 amino acids with seven to ten potential transmembrane domains and additional motifs. The blm3 null mutation was created by gene replacement, and found not to be essential for growth in the absence of the bleomycin-phleomycin antibiotics. Sequence analyses suggest the Blm3p could be a potential member of the major facilitator superfamily (MFS) of permeases. Northern dot blot analyses using a human RNA master tissue blot containing RNA from fifty different fetal and adult tissues revealed sequence homology in adult tissues to BLM3, but no sequence homology in fetal tissues. The function of the Blm3p is presently unknown. We propose several functions for the Blm3p in protecting cells against oxidative agents, including roles in detoxification, transport and defending against DNA damage.

Biochemical and cellular effects of c-Src kinase-selective pyrido[2, 3-d]pyrimidine tyrosine kinase inhibitors.

Increased expression or activity of c-Src tyrosine kinase has been associated with the transformed phenotype in tumor cells and with progression of neoplastic disease. A number of pyrido[2, 3-d]pyrimidines have been characterized biochemically and in cells as part of an assessment of their potential as anti-tumor agents. The compounds were ATP-competitive inhibitors of c-Src kinase with IC(50) values < 10 nM and from 6 to >100-fold selectivity for c-Src tyrosine kinase relative to basic fibroblast growth factor receptor (bFGFr) tyrosine kinase, platelet-derived growth factor receptor (PDGFr) tyrosine kinase, and epidermal growth factor receptor (EGFr) tyrosine kinase. The compounds yielded IC(50) values < 5 nM against Lck. Human colon tumor cell growth in culture was inhibited, as was colony formation in soft agar at concentrations < 1 microM. Phosphorylation of the c-Src cellular substrates paxillin, p130(cas), and Stat3 was also inhibited at concentrations < 1 microM. Autophosphorylation of EGFr tyrosine kinase or PDGFr tyrosine kinase was not inhibited by c-Src inhibitors, thus showing the selective nature of the compounds in cells. In a mitogenesis assay measuring thymidine incorporation stimulated by specific mitogens, the c-Src tyrosine kinase inhibitors reduced incorporated thymidine in a manner consistent with previously reported roles of c-Src in mitogenic signaling. Progression through the cell cycle was inhibited at G(2)/M in human colon tumor cells treated with two of the c-Src-selective compounds, which is also consistent with earlier reports describing a requirement for active c-Src tyrosine kinase for G(2) to M phase progression. The compounds described here are selective inhibitors of c-Src tyrosine kinase and have antiproliferative effects in tumor cells consistent with inhibition of c-Src.

DNA damage-inducible and RAD52-independent repair of DNA double-strand breaks in Saccharomyces cerevisiae.

Chromosomal repair was studied in stationary-phase Saccharomyces cerevisiae, including rad52/rad52 mutant strains deficient in repairing double-strand breaks (DSBs) by homologous recombination. Mutant strains suffered more chromosomal fragmentation than RAD52/RAD52 strains after treatments with cobalt-60 gamma irradiation or radiomimetic bleomycin, except after high bleomycin doses when chromosomes from rad52/rad52 strains contained fewer DSBs than chromosomes from RAD52/RAD52 strains. DNAs from both genotypes exhibited quick rejoining following gamma irradiation and sedimentation in isokinetic alkaline sucrose gradients, but only chromosomes from RAD52/RAD52 strains exhibited slower rejoining (10 min to 4 hr in growth medium). Chromosomal DSBs introduced by gamma irradiation and bleomycin were analyzed after pulsed-field gel electrophoresis. After equitoxic damage by both DNA-damaging agents, chromosomes in rad52/rad52 cells were reconstructed under nongrowth conditions [liquid holding (LH)]. Up to 100% of DSBs were eliminated and survival increased in RAD52/RAD52 and rad52/rad52 strains. After low doses, chromosomes were sometimes degraded and reconstructed during LH. Chromosomal reconstruction in rad52/rad52 strains was dose dependent after gamma irradiation, but greater after high, rather than low, bleomycin doses with or without LH. These results suggest that a threshold of DSBs is the requisite signal for DNA-damage-inducible repair, and that nonhomologous end-joining repair or another repair function is a dominant mechanism in S. cerevisiae when homologous recombination is impaired.

A spheroplast rate assay for determination of cell wall integrity in yeast.

The rate of formation of spheroplasts of yeast can be used as an assay to study the structural integrity of cell walls. Lysis can be measured spectrophotometrically in hypotonic solution in the presence of Zymolyase, a mixture of cell wall-digesting enzymes. The optical density of the cell suspension decreases as the cells lyse. We optimized this assay with respect to enzyme concentration, temperature, pH, and growth conditions for several strains of Saccharomyces cerevisiae. The level of variability (standard deviation) was 1-5% between trials where the replications were performed on the same culture using enzyme prepared from the same lot, and 5-15% for different cultures of the same strain. This assay can quantitate differences in cell wall structure (1) between exponentially growing and stationary phase cells, (2) among different S. cerevisiae strains, (3) between S. cerevisiae and Candida albicans, (4) between parental and mutated lines, and (5) between drug- or chemically-treated cells and controls.

Tyrosine kinase inhibitors. 6. Structure-activity relationships among N- and 3-substituted 2,2'-diselenobis(1H-indoles) for inhibition of protein tyrosine kinases and comparative in vitro and in vivo studies against selected sulfur congeners.

A small series of 2,2'-diselenobis(1H-indoles) was synthesized as redox-modified congeners of our earlier reported 2,2'-dithiobis(1H-indole) series. Utilizing chemistry similar to that developed earlier for the disulfur series, compounds were made from 2-halogeno-3-indolecarboxylic acid precursors bearing various polar functionality at the C-3 position and small alkyl substituents at the N-1 position of the indole nucleus. Additional compounds were derived from (R)- or (S)-tryptophan via a novel application of diselenium dichloride as an electrophilic source of diselenium, and a much improved process to a 2,2'-dithiobis(1H-indole) congener was developed utilizing disulfur dichloride as a source of disulfur. Against isolated epidermal growth factor receptor (EGFr), platelet-derived growth factor receptor (PDGFr), and v-src tyrosine kinases, compounds in this series displayed broad inhibitory activity with IC50 = 0.9 to > 100 microM vs EGFr, 3.4 to > 50 microM vs PDGFr, and 0.4-6.7 microM vs v-src. In general, compounds derived from tryptophan displayed the greatest potency against EGFr and those from 2-halogeno-3-indolecarboxylic acids greater potency against PDGFr and v-src. Enzyme kinetics studies showed that both classes of compounds display primarily noncompetitive inhibition with respect to either ATP or peptide substrate. The sulfhydryl reducing agent dithiothreitol (DTT) caused a general decrease in inhibition of the EGFr and v-src tyrosine kinases by both the diselenium and disulfur series with the reversal of enzyme inhibition occurring less readily within the diselenium series. In whole cell studies, compounds of this class were growth inhibitory against Swiss 3T3 mouse fibroblasts with IC50 values from 0.5 to 19.5 microM, and the observed SAR was different from that of the 2,2'-dithiobis(1H-indoles). A comparative study in the same cell line on the effects of the 2,2'-diselenobis(1H-indole) derived from (R)-tryptophan vs its disulfur congener on growth factor mediated tyrosine phosphorylation showed that this compound significantly inhibited EGFr and PDGFr (in response to its ligand) autophosphorylation with complete suppression at 25 and 5 microM, respectively. Tyrosine phosphorylation of an 85 kDa protein typically phosphorylated in response to bFGF was also exquisitely sensitive to this compound, and it displayed inhibitory effects on DNA, RNA, and protein synthesis at submicromolar concentrations. The disulfur congener exhibited a qualitatively similar pattern; however, its potency was 10-fold less. This same diselenium/disulfur pair was evaluated in vivo against the B16 melanoma, colon carcinoma 26, and M5076 sarcoma murine tumors, and the A431 epidermoid, and C6 glioma human tumor xenografts. At maximum tolerated doses (1.8 and 5.0 mg/kg/injection, respectively), neither the diselenium nor disulfur congener was effective against the C6 glioma when administered intraperitoneally on a d1-9 schedule. Studies were also carried out against the A431 epidermoid xenograft to evaluate the same pair of compounds via continuous subcutaneous infusion from Alzet miniosmotic pumps. The maximum dose that could be administered daily was limited by compound solubility. Neither compound produced an antitumor effect in a 7-day continuous infusion study. In the 27-day study, the disulfur compound was inactive whereas the diselenium compound produced a 10.8-day growth delay without appreciable treatment related weight loss. The in vitro and in vivo findings offer a mechanistic rationale as to why the 2,2'-diselenobis(1H-indoles) are more potent inhibitors than their disulfur congeners.

Cloning, characterization and identification of the gene encoding phosphatidylinositol 4-kinase.

The vast majority of AIDS-related deaths are associated with opportunistic infections. For fungal infections, there are few effective antifungals, particularly for systemic use. The discovery that very low doses of the bleomycin family of anticancer chemical congeners compromise the integrity of fungal cell walls led to our approach to identify genes that complement-cell wall defects, and develop methods to facilitate the identification of new antifungals targeted to fungal cell walls. This report describes one of the genes cloned by complementation of the blm1-1 mutation of S. cerevisiae using a YCp50-based yeast genomic library. Characterization and identification of the gene were carried out using drug screening tests, Southern hybridization analyses, DNA sequencing and DNA sequence similarity searches in databases. The gene STT4, is essential for viability and encodes a phosphatidylinositol 4-kinase that plays an important role in the phosphatidylinositol-mediated signal transduction pathway required for cell wall integrity. Like blm1-1 mutant strains, stt4 cells arrest mostly in the G2/M phase of the cell cycle. Further studies using this approach should help us understand the role of PI4-K in maintaining fungal cell-wall integrity, identify additional genes affecting potential target structures in cell walls of opportunistic fungal pathogens in AIDS patients, and assist in drug discovery and antifungal drug design.

The effects of volunteering on the young volunteer.

This article reviews some of the best researched volunteer service programs for adolescents and addresses three major questions: 1) What do existing data tell us about the effectiveness of community volunteer service programs in positively influencing the lives of the participants? 2) What do we know about why such programs work? 3) What are the most promising directions for future research and programming efforts to pursue? The review suggests that diverse, successful volunteer programs for adolescents, along with school-based support, are related to improvements in both the academic and social arenas. Specifically, volunteering relates to reduced rates of course failure, suspension from school, and school dropout, and improvement in reading grades; to a reduction in teen pregnancy; and to improved self-concept and attitudes toward society. The conditions under which the volunteering occurs, such as number of hours and the type of volunteer work, seem in some cases to be important to these outcomes, as does the age of the student volunteer; however, much is yet to be understood about these factors.

Oxidative cell wall damage mediated by bleomycin-Fe(II) in Saccharomyces cerevisiae.

Bleomycin mediates cell wall damage in the yeast Saccharomyces cerevisiae. Bleomycin treatments in the presence of Fe(II) increased the rate of spheroplast formation by lytic enzymes by 5- to 40-fold. Neither Fe(III) nor other tested ions caused significant cell wall damage in the presence of bleomycin. The effect of bleomycin-Fe(II) on the cell wall mimicked the characteristics of bleomycin-Fe(II)-mediated DNA damage in dependence on aeration, inhibition by ascorbate, and potentiation by submillimolar concentrations of sodium phosphate. Bleomycin-mediated cell wall damage was time and dose dependent, with incubations as short as 20 min and drug concentrations as low as 3.3 x 10(-7)M causing measurable cell wall damage in strain CM1069-40. These times and concentrations are within the range of effectiveness for bleomycin-mediated DNA damage and for the cytotoxicity of the drug. Although Fe(III) was inactive with bleomycin and O2, the bleomycin-Fe(III) complex damaged walls and lysed cells in the presence of H2O2. H2O2 causes similar activation of bleomycin-Fe(III) in assays of DNA scission. These results suggest that an activated bleomycin-Fe-O2 complex disrupts essential cell wall polymers in a manner analogous to bleomycin-mediated cleavage of DNA.

A mutant approach and molecular strategy to study fungal cell walls.

The current study describes recombinant plasmids which complement the hypersusceptibility to killing bleomycin of blm1-1 mutant cells of Saccharomyces cerevisiae, and a strategy developed and used to recover active clones from a stable yeast genomic library. The resistance of a spontaneous revertant isolated from the original blm1-1 mutant strain and of mutant cells transformed with each of several recombinant plasmids which complemented the recessive blm1-1 mutation was comparable to the resistance of the parental (non-mutant) strain from which the original blm1-1 mutant was derived. The strategy for cloning S. cerevisiae DNA was based on complementation and in situ hybridization. This strategy employed 32P-labelled 6.6-kb BamHI and 3.8-kb BamHI-ClaI probes from a cloned DNA fragment to recover clones which either fully or partially complemented the hypersensitivity of mutant cells to killing by bleomycin. This method considerably reduced the time and effort required to recover biologically active clones from a genomic library.

Genetic changes and bioassays in bleomycin- and phleomycin-treated cells, and their relationship to chromosomal breaks.

The recombinogenicity of damaged chromosomes in diploid Saccharomyces cerevisiae cells treated with bleomycin and structurally related phleomycin was measured, along with aneuploidy and mutation events. Phleomycin was substantially (up to 26-fold) more effective than bleomycin in producing genetic changes at all concentrations, even when colony-forming abilities of cells growing in the presence of bleomycin or phleomycin were similar. These results suggest that the DNA lesions produced by the two structurally related analogs could differ in their nature or frequency, or could be processed differently by the cells. Bioassays were developed and used to compare the cytotoxicities of freshly dissolved bleomycin and phleomycin with the cytotoxicities of lysates prepared from bleomycin- and phleomycin-treated cells. Unexpectedly, lysates prepared from bleomycin-treated cells were 1.5-3.5 times more cytotoxic than freshly dissolved bleomycin after 45-min treatments (3-33 x 10(-6) M). In contrast, lysates prepared from phleomycin-treated cells were 3-38 times less cytotoxic than freshly dissolved phleomycin (0.5-6.4 x 10(-6) M). Cytotoxicities of all lysates were higher after 36-h treatments than after 45-min treatments. At 3.3 x 10(-6) M, this increase was eightfold for bleomycin and 15-fold for phleomycin. Nevertheless, lysates from phleomycin-treated cells were considerably more cytotoxic than lysates from bleomycin-treated cells or freshly prepared bleomycin, consistent with the higher effectiveness of phleomycin than bleomycin in producing chromosomal breaks, genetic changes, and cell killing.

Potentiation of bleomycin cytotoxicity in Saccharomyces cerevisiae.

Lesions introduced into cellular DNAs prelabeled with [2-14C]thymidine or [6-3H]thymidine, as well as cell killing, were inhibited by the presence of EDTA during 20-min reactions of Saccharomyces cerevisiae cells with the low-molecular-weight bleomycin family of anticancer antibiotics. In contrast, the level of killing by low concentrations of bleomycin was higher among cells which had grown for three generations in defined synthetic complete medium supplemented with ferrous sulfate than among cells grown without iron supplementation. In S. cerevisiae, the uptake of iron is facilitated by a plasma membrane ferric reductase activity and a high-affinity (Km = 5 x 10(-6) M) ferrous uptake system. Lethal effects of 1.3 x 10(-6) M bleomycin increased approximately 50% with 10(-5) M Fe(II), nearly twofold with 10(-4) M Fe(II), and 2.8 times with 10(-3) M Fe(II). Thus, iron preloading is a new experimental approach to increasing and studying the effects of the glycopeptides on cellular DNAs and other cellular targets. This approach could also be used for studying and better understanding DNA repair genes and could serve as a model for studies of redox active chemicals in biological systems.

Bleomycin affects cell wall anchorage of mannoproteins in Saccharomyces cerevisiae.

Bleomycin induces strand breakage in DNA through disruption of glycosidic linkages. We investigated the ability of bleomycin to damage yeast cell walls, which are composed primarily of carbohydrate. Bleomycin treatment of intact yeast cells facilitated enzymatic conversion of yeasts to spheroplasts. Bleomycin treatment also altered anchorage of mannoproteins to the cell wall matrix in intact cells or isolated cell walls. Cell surface mannoproteins were labelled with 125I, and their solubilization was monitored. Seventeen hour treatments with bleomycin released some of the label directly into treatment supernatants and facilitated extraction of mannoproteins by dithiothreitol and lytic enzymes. Bleomycin treatments as short as 10 min caused changes in extraction of mannoproteins from intact cells. Specifically, cell wall anchorage of several mannoproteins was affected by the drug. There were drug-induced changes in extractability of mannoproteins with apparent molecular weights of 96,000, 80,000, 61,000, 41,000, 31,500, and 21,000 (determined after deglycosylation with endo-N-acetylglucosaminidase H). The similarity of results obtained in the presence and absence of cycloheximide, the appearance of cell wall effects after only 10 min of treatment, and the similarity of effects in intact cells and isolated cell walls are consistent with direct drug-induced damage and inconsistent with a mechanism dependent on expression of bleomycin-damaged genes or other intracellular mediators. The results are consistent with bleomycin-mediated increases in cell wall permeability through disruption of glycosidic cross-linking structures in the cell wall.

Lesions and preferential initial localization of [S-methyl-3H]bleomycin A2 on Saccharomyces cerevisiae cell walls and membranes.

Extensive lesions were produced in cell walls of Saccharomyces cerevisiae by the bleomycin family of anticancer antibiotics (30 min to 4 h). Electron micrographs revealed that the alterations were most frequently large breaks and small interruptions or holes in cell walls, which sometimes extended into cell membranes. Large portions of cell walls were sometimes lost. Cell walls were frequently ruptured in one or more positions. More than 75% of bud scar regions in single-plane sections and all bud scars in serial sections exhibited many interruptions and breaks after 3 or 4 h of treatment. The discovery of extensive damage to cell walls was consistent with the preferential (approximately 70%) association of radiolabeled bleomycin with cell walls and perimeters of bud scar regions after short exposures (30 min). After longer exposures, the distribution of silver grains changed from a predominant association with cell walls (30 min) to an increased association with the cell cytoplasm (1 to 4 h). This correlated with increased ultrastructural damage, since damage to cell walls was generally more frequent and more severe with increasing length of treatment (30 min to 4 h) or dose (25 to 100 micrograms/ml). Although DNA lesions are believed to be the lethal properties of bleomycins, the lesions produced in cell walls are also lethal properties of the antibiotics. The distributions of lesions on cell walls suggested a generalized interaction of the antibiotic with a cell wall component. These results led us to hypothesize a mechanism of effective antifungal action for the bleomycin family of antibiotics.

Further characterizations of bleomycin-sensitive (blm) mutants of Saccharomyces cerevisiae with implications for a radiomimetic model.

Direct selection for 12 mutations (blm) conferring hypersensitivities to lethal effects of bleomycins in Saccharomyces cerevisiae resulted in mutants exhibiting cross-hypersensitivity to ionizing radiation and hydrogen peroxide. Remaining mutations did not confer cross-hypersensitivity to radiation. All blm mutations were recessive, except codominant blm3-1, and were assigned to seven complementation groups.

Preclinical antitumor activity of CI-973,[SP-4-3-(R)]-[1,1- cyclobutanedicarboxylato(2-)](2 methyl-1,4-butane-diamine-N,N')platinum.

The antitumor activity of [SP-4-3-(R)]-[1,1-cyclobutanedicarboxylato-(2-)](2 methyl-1,4-butanediamine-N,N')platinum (CI-973) was characterized in a number of preclinical model systems. CI-973 retained substantial activity in cisplatin resistant murine leukemia cell lines, in vitro and in vivo; in L1210 leukemia resistant to cisplatin in vivo, CI-973 retained as much activity as was found in animals bearing sensitive L1210 leukemia. When compared in five solid murine tumors in vivo, both CI-973 and cisplatin were approximately equivalent in activity. In one human colon tumor and one human non-small cell lung carcinoma tested as xenografts in immunodeficient mice, cisplatin and CI-973 were ineffective. In two other human non-small cell lung carcinomas tested in the same fashion, cisplatin did possess activity. CI-973 has entered phase I clinical trials.

Degradation of DNA and structure-activity relationship between bleomycins A2 and B2 in the absence of DNA repair.

The contribution of DNA repair to the net number of DNA breaks produced during chemical degradation of DNA was determined by using temperature-sensitive mutant cells deficient in ATP-dependent DNA ligase [poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase, EC 6.5.1.1]. In a very sensitive assay for determining lesions introduced into Saccharomyces cerevisiae DNAs, 2-14C- and 6-3H-prelabeled DNAs from ligase-proficient and ligase-deficient cells were sedimented together through precalibrated, isokinetic alkaline sucrose gradients. DNA ligation was slower after chemical degradation of DNA by bleomycin than after gamma irradiation. DNA breaks increased approximately linearly with drug concentrations, and were approximately equivalent for ligase-proficient and ligase-deficient cells. These results were unexpected because ligase-deficient, but not ligase-proficient, cells lacked the capacity to eliminate DNA breaks produced by bleomycin. The results indicated that DNA repair did not occur during the chemical degradation of DNA under the experimental conditions. Bleomycin B2 produced considerably more DNA breaks than bleomycin A2 over a range of concentrations in ligase-proficient cells, which tolerated higher numbers of DNA breaks in general than ligase-deficient cells. The chemical analogues are structurally identical except for their cationic C-terminal amine. The actual number of DNA breaks produced by bleomycin A2 or bleomycin B2, and not the concentration of bleomycin A2 or bleomycin B2 per se, determined the amount of cell killing. DNA repair is critical in quantitating DNA breaks produced by chemicals, but was ruled out as a factor in the higher DNA breakage by bleomycin B2 than bleomycin A2.