IFNγ production by NK cells from HLA-sensitized patients after in vitro exposure to allo-antigens.

Using a novel cytokine flow cytometry test (allo-CFC), we have previously shown that incubation of allogeneic cells with peripheral blood from highly-HLA sensitized (HS) patients results in reproducible gamma-interferon (IFNγ production in CD3(-) cells, and high (+) allo-CFC levels correlated with risk for antibody-mediated rejection (AMR). Here we report on identification of the cells and mechanisms responsible. The allo-CFC with/without modification was performed using blood from HS or normal individuals. IFNγ producing cells were CD3(-)/CD19(-), but CD3(-)/CD56(+). In vitro and in vivo B cell-depletion did not affect IFNγ production, demonstrating NK cells as the cells responsible for IFNγ production. NK cells from allo-CFC(+) or (-) individuals released significant amounts of IFNγ against target cells treated with serum from allo-CFC(+) individuals, but not allo-CFC(-) individuals. IFNγ release was abrogated by protein A/G treatment of the pretreated target cells, suggesting mediation by antibodies via FcγRIIIa (CD16). In conclusion, NK cell IFNγ release after allo-antigen exposure is mediated primarily through antibody-dependent cellular cytotoxicity (ADCC)-like mechanisms, suggesting that NK cells may be partially responsible for graft injury during AMR including C4d(-) AMR via ADCC, and could be a potential target for modification of this process.

Ultraviolet irradiation induces BRCA2 protein depletion through a p53-independent and protein synthesis-dependent pathway.

It has been suggested that BRCA2, the protein product of the breast cancer susceptibility gene BRCA2, is involved in DNA damage repair. It is therefore likely that BRCA2 plays a role in a signaling pathway induced by DNA-damaging agents. To test this possibility, we examined the alteration of the BRCA2 protein level in human cell lines after UV irradiation. We found that UV irradiation down-regulated BRCA2 in a dose-dependent manner in all cell lines tested. The down-regulation of BRCA2 occurred soon (within 4 h) after UV treatment. Surprisingly, down-regulation of BRCA2 by UV does not require functional p53, which has been suggested to be required for the down-regulation of BRCA1 and BRCA2 mRNAs by DNA-damaging agents. Moreover, the proteosome- and calpain-mediated protein degradation pathways do not have an important role in the UV-induced BRCA2 depletion. However, blocking protein synthesis temporally inhibited the depletion of BRCA2 and BRCA1 in some cell lines. Ectopic expression of BRCA2 in cells increased resistance of cells to high-dose UV irradiation. These results demonstrate that BRCA2 is involved in a DNA-damaging signaling pathway induced by UV radiation and that expression of BRCA2 can protect cells from UV-mediated cell death.

Cyclobutane pyrimidine dimers and bulky chemical DNA adducts are efficiently repaired in both strands of either a transcriptionally active or promoter-deleted APRT gene.

Both prokaryotic and eukaryotic cells have the capacity to repair DNA damage preferentially in the transcribed strand of actively expressed genes. However, we have found that several types of DNA damage, including cyclobutane pyrimidine dimers (CPDs) are repaired with equal efficiency in both the transcribed and nontranscribed strands of the adenine phosphoribosyltransferase (APRT) gene in Chinese hamster ovary cells. We further found that, in two mutant cell lines in which the entire APRT promoter region has been deleted, CPDs are still efficiently repaired in both strands of the promoterless APRT gene, even though neither strand appears to be transcribed. These results suggest that efficient repair of both strands at this locus does not require transcription of the APRT gene. We have also mapped CPD repair in exon 3 of the APRT gene in each cell line at single nucleotide resolution. Again, we found similar rates of CPD repair in both strands of the APRT gene domain in both APRT promoter-deletion mutants and their parental cell line. Our findings suggest that current models of transcription-coupled repair and global genomic repair may underestimate the importance of factors other than transcription in governing the efficiency of nucleotide excision repair.

Slow repair of bulky DNA adducts along the nontranscribed strand of the human p53 gene may explain the strand bias of transversion mutations in cancers.

Using UvrABC incision in combination with ligation-mediated PCR (LMPCR) we have previously shown that benzo(a)pyrene diol epoxide (BPDE) adduct formation along the nontranscribed strand of the human p53 gene is highly selective; the preferential binding sites coincide with the major mutation hotspots found in human lung cancers. Both sequence-dependent adduct formation and repair may contribute to these mutation hotspots in tumor tissues. To test this possibility, we have extended our previous studies by mapping the BPDE adduct distribution in the transcribed strand of the p53 gene and quantifying the rates of repair for individual damaged bases in exons 5, 7, and 8 for both DNA strands of this gene in normal human fibroblasts. We found that: (i) on both strands, BPDE adducts preferentially form at CpG sequences, and (ii) repair of BPDE adducts in the transcribed DNA strand is consistently faster than repair of adducts in the nontranscribed strand, while repair at the major damage hotspots (guanines at codons 157, 248 and 273) in the nontranscribed strand is two to four times slower than repair at other damage sites. These results strongly suggest that both preferential adduct formation and slow repair lead to hotspots for mutations at codons 157, 248 and 273, and that the strand bias of bulky adduct repair is primarily responsible for the strand bias of G to T transversion mutations observed in the p53 gene in human cancers.

Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in P53.

Cigarette smoke carcinogens such as benzo[a]pyrene are implicated in the development of lung cancer. The distribution of benzo[a]pyrene diol epoxide (BPDE) adducts along exons of the P53 gene in BPDE-treated HeLa cells and bronchial epithelial cells was mapped at nucleotide resolution. Strong and selective adduct formation occurred at guanine positions in codons 157, 248, and 273. These same positions are the major mutational hotspots in human lung cancers. Thus, targeted adduct formation rather than phenotypic selection appears to shape the P53 mutational spectrum in lung cancer. These results provide a direct etiological link between a defined chemical carcinogen and human cancer.

Sequence preference of 7,12-dimethylbenz[a]anthracene-syn-diol epoxide-DNA binding in the mouse H-ras gene detected by UvrABC nucleases.

We have found that 7,12-dimethylbenz[a]anthracene-syn-diol epoxide (syn-DMBADE)-modified DNA fragments are sensitive to UvrABC incision. The incisions occur mainly seven bases 5' and four bases 3' of a syn-DMBADE-modified adenine or guanine residue. The kinetics of UvrABC incision at different sequences in a DNA fragment are the same, and the extent of UvrABC incision is proportional to the syn-DMBADE concentration. On the basis of these results, we have concluded that UvrABC incision on syn-DMBADE-DNA adducts is independent of DNA sequence and is quantitative. Using the UvrABC incision method, we have analyzed the syn-DMBADE-DNA binding spectrum in several defined DNA fragments, including the first two exons of the mouse H-ras gene. We have found that both guanine and adenine residues in codons 12, 13, and 61 of the H-ras gene are strong syn-DMBADE binding sites. These results suggest that the initial binding of DMBADE may greatly contribute to the frequency of H-ras mutations. Results from dinucleotide binding analysis indicate that the 5'-nearest neighbor displays a greater effect on syn-DMBADE-DNA binding than the 3'-nearest neighbor.

Repair of benzo(a)pyrene diol epoxide- and UV-induced DNA damage in dihydrofolate reductase and adenine phosphoribosyltransferase genes of CHO cells.

Using Uvr proteins we have quantified benzo(a)pyrene diol epoxide (BPDE)-DNA adduct formation and repair at the dihydrofolate reductase (DHFR) and adenine phosphoribosyltransferase (APRT) genes in two Chinese hamster ovary cell lines: B-11 cells, which are 50-fold amplified for DHFR, and AT3-2 cells, which are diploid for DHFR. We have found that: 1) BPDE-DNA adduct formation in different regions of the DHFR gene is proportional to the concentration of BPDE. 2) There is no significant difference in the repair of BPDE-DNA adducts between the coding and noncoding regions in either amplified or nonamplified DHFR gene domains. 3) Repair in the nonamplified DHFR gene is more efficient (30-40%) than in the amplified DHFR genes. 4) There are no significant differences of repair in the transcribed or nontranscribed strands of the DHFR gene. 5) BPDE-DNA adduct formation and repair in the APRT gene in B-11 and AT3-2 cells are the same. These results contrast those for the repair of cyclobutane pyrimidine dimers, which occurs preferentially in the transcribed strand of the DHFR gene and in which gene amplification appears to play no role.

Formation and repair of antitumor antibiotic CC-1065-induced DNA adducts in the adenine phosphoribosyltransferase and amplified dihydrofolate reductase genes of Chinese hamster ovary cells.

CC-1065 is a potent antitumor antibiotic which bonds to duplex DNA specifically; the biological effects of the drug are presumably the consequences of its DNA interactions. In order to investigate the factors which may affect drug-DNA bonding in cells, a method using a thermal-alkaline treatment to induce phosphodiester bond breakage at the drug-DNA bonding sites and Southern DNA transfer-hybridization to quantify drug-DNA bonding at defined sequences in drug-treated cultured mammalian cells was developed. We have found that in vivo, in cultured Chinese hamster ovary (CHO) cells, CC-1065 bonds twice as efficiently in the highly amplified dihydrofolate reductase (DHFR) gene domains as in the nonamplified adenine phosphoribosyltransferase (APRT) gene domain. However, in vitro, in purified CHO cellular DNA, CC-1065 bonds equally to both the DHFR and APRT genes. We observed a significant degree of "gene-specific" preferential repair for drug-DNA adducts in the amplified DHFR gene domains, and it appears that this "gene-specific" repair reflects "transcribed-strand specific" repair. These results suggest that DNA amplification may affect drug-DNA adduct formation and transcription may affect its repair.

Genetic control of capsid length in bacteriophage T4: clustering of ptg mutations in gene 23.

Fifty-two new bacteriophage T4 ptg mutations have been isolated by selecting for the giant-capsid phenotype they display. Genetic mapping placed all of them at eight sites, all located in gene 23. These sites were clustered in three locations, one near amber B17 (gene 23 nucleotide [NT] 268), another centrally placed between amE506 (NT 706) and amE1270 (NT 925), and the third between amC208 (NT 1297) and amE1236 (NT 1489). The lack of a selective system for identifying recombinant genotypes when dealing with the very close linkages found within these clusters opens the possibility that more than eight sites are represented in this set of mutations. Since one site was represented by only one mutation, it seems likely that further searching might uncover additional sites. It is suggested that the clustering of mutations observed here identifies regions of the gene 23 product that play a role in regulating the capsid length of T4.

Genetic control of capsid length in bacteriophage T4: phenotypes displayed by ptg mutants.

The phenotypic characteristics of 26 ptg mutations in T4 gene 23 are described. All were located in three tight clusters in that gene and, by definition of ptg mutations, all produced giant phage. Intermediate petite phage, which invariably made up a substantial fraction of the progeny of these mutants, appeared to be a unique product of gene 23 mutations. Isometric petite phage were produced in significant numbers by strains with mutations at only 4 of the 10 sites identified with the PTG phenotype. The data presented indicate that there was little if any variation in the lengths of the normal, the intermediate petite, and the isometric petite classes. The frequencies of those capsid types were fairly specific for the individual mutations. The giant capsids that resulted from ptg mutations also had characteristic length distributions, of which three types were distinguished. These highly specific effects of gene 23 ptg mutations on capsid length regulation of T4 imply that the product of gene 23, gp23, plays a significant role in controlling the length of its capsid. The restrictions these observations place on a model for T4 capsid length regulation are discussed briefly.