Renal Allograft Survival in Nonhuman Primates Infused With Donor Antigen-Pulsed Autologous Regulatory Dendritic Cells.

Systemic administration of autologous regulatory dendritic cells (DCreg; unpulsed or pulsed with donor antigen [Ag]), prolongs allograft survival and promotes transplant tolerance in rodents. Here, we demonstrate that nonhuman primate (NHP) monocyte-derived DCreg preloaded with cell membrane vesicles from allogeneic peripheral blood mononuclear cells induce T cell hyporesponsiveness to donor alloantigen (alloAg) in vitro. These donor alloAg-pulsed autologous DCreg (1.4-3.6 × 106 /kg) were administered intravenously, 1 day before MHC-mismatched renal transplantation to rhesus monkeys treated with costimulation blockade (cytotoxic T lymphocyte Ag 4 immunoglobulin [CTLA4] Ig) and tapered rapamycin. Prolongation of graft median survival time from 39.5 days (no DCreg infusion; n = 6 historical controls) and 29 days with control unpulsed DCreg (n = 2), to 56 days with donor Ag-pulsed DCreg (n = 5) was associated with evidence of modulated host CD4+ and CD8+ T cell responses to donor Ag and attenuation of systemic IL-17 production. Circulating anti-donor antibody (Ab) was not detected until CTLA4 Ig withdrawal. One monkey treated with donor Ag-pulsed DCreg rejected its graft in association with progressively elevated anti-donor Ab, 525 days posttransplant (160 days after withdrawal of immunosuppression). These findings indicate a modest but not statistically significant beneficial effect of donor Ag-pulsed autologous DCreg infusion on NHP graft survival when administered with a minimal immunosuppressive drug regimen.

Regulatory T Cell Infusion Can Enhance Memory T Cell and Alloantibody Responses in Lymphodepleted Nonhuman Primate Heart Allograft Recipients.

The ability of regulatory T cells (Treg) to prolong allograft survival and promote transplant tolerance in lymphodepleted rodents is well established. Few studies, however, have addressed the therapeutic potential of adoptively transferred, CD4(+) CD25(+) CD127(-) Foxp3(+) (Treg) in clinically relevant large animal models. We infused ex vivo-expanded, functionally stable, nonselected Treg (up to a maximum cumulative dose of 1.87 billion cells) into antithymocyte globulin-lymphodepleted, MHC-mismatched cynomolgus monkey heart graft recipients before homeostatic recovery of effector T cells. The monkeys also received tacrolimus, anti-interleukin-6 receptor monoclonal antibodies and tapered rapamycin maintenance therapy. Treg administration in single or multiple doses during the early postsurgical period (up to 1 month posttransplantation), when host T cells were profoundly depleted, resulted in inferior graft function compared with controls. This was accompanied by increased incidences of effector memory T cells, enhanced interferon-γ production by host CD8(+) T cells, elevated levels of proinflammatory cytokines, and antidonor alloantibodies. The findings caution against infusion of Treg during the early posttransplantation period after lymphodepletion. Despite marked but transient increases in Treg relative to endogenous effector T cells and use of reputed "Treg-friendly" agents, the host environment/immune effector mechanisms instigated under these conditions can perturb rather than favor the potential therapeutic efficacy of adoptively transferred Treg.

Sequential monitoring and stability of ex vivo-expanded autologous and nonautologous regulatory T cells following infusion in nonhuman primates.

Ex vivo-expanded cynomolgus monkey CD4(+)CD25(+)CD127(-) regulatory T cells (Treg) maintained Foxp3 demethylation status at the Treg-specific demethylation region, and potently suppressed T cell proliferation through three rounds of expansion. When carboxyfluorescein succinimidyl ester- or violet proliferation dye 450-labeled autologous (auto) and nonautologous (non-auto)-expanded Treg were infused into monkeys, the number of labeled auto-Treg in peripheral blood declined rapidly during the first week, but persisted at low levels in both normal and anti-thymocyte globulin plus rapamycin-treated (immunosuppressed; IS) animals for at least 3 weeks. By contrast, MHC-mismatched non-auto-Treg could not be detected in normal monkey blood or in blood of two out of the three IS monkeys by day 6 postinfusion. They were also more difficult to detect than auto-Treg in peripheral lymphoid tissue. Both auto- and non-auto-Treg maintained Ki67 expression early after infusion. Sequential monitoring revealed that adoptively transferred auto-Treg maintained similarly high levels of Foxp3 and CD25 and low CD127 compared with endogenous Treg, although Foxp3 staining diminished over time in these nontransplanted recipients. Thus, infused ex vivo-expanded auto-Treg persist longer than MHC-mismatched non-auto-Treg in blood of nonhuman primates and can be detected in secondary lymphoid tissue. Host lymphodepletion and rapamycin administration did not consistently prolong the persistence of non-auto-Treg in these sites.

Combined in vivo and in silico investigations of activation of glycolysis in contracting skeletal muscle.

The hypothesis was tested that the variation of in vivo glycolytic flux with contraction frequency in skeletal muscle can be qualitatively and quantitatively explained by calcium-calmodulin activation of phosphofructokinase (PFK-1). Ischemic rat tibialis anterior muscle was electrically stimulated at frequencies between 0 and 80 Hz to covary the ATP turnover rate and calcium concentration in the tissue. Estimates of in vivo glycolytic rates and cellular free energetic states were derived from dynamic changes in intramuscular pH and phosphocreatine content, respectively, determined by phosphorus magnetic resonance spectroscopy ((31)P-MRS). Computational modeling was applied to relate these empirical observations to understanding of the biochemistry of muscle glycolysis. Hereto, the kinetic model of PFK activity in a previously reported mathematical model of the glycolytic pathway (Vinnakota KC, Rusk J, Palmer L, Shankland E, Kushmerick MJ. J Physiol 588: 1961-1983, 2010) was adapted to contain a calcium-calmodulin binding sensitivity. The two main results were introduction of regulation of PFK-1 activity by binding of a calcium-calmodulin complex in combination with activation by increased concentrations of AMP and ADP was essential to qualitatively and quantitatively explain the experimental observations. Secondly, the model predicted that shutdown of glycolytic ATP production flux in muscle postexercise may lag behind deactivation of PFK-1 (timescales: 5-10 s vs. 100-200 ms, respectively) as a result of accumulation of glycolytic intermediates downstream of PFK during contractions.

BRCA2 mutations in primary breast and ovarian cancers.

The second hereditary breast cancer gene, BRCA2, was recently isolated. Germline mutations of this gene predispose carriers to breast cancer, and, to a lesser extent, ovarian cancer. Loss of heterozygosity (LOH) at the BRCA2 locus has been observed in 30-40% of sporadic breast and ovarian tumours, implying that BRCA2 may act as a tumour suppressor gene in a proportion of sporadic cases. To define the role of BRCA2 in sporadic breast and ovarian cancer, we screened the entire gene for mutations using a combination of techniques in 70 primary breast carcinomas and in 55 primary epithelial ovarian carcinomas. Our analysis revealed alterations in 2/70 breast tumours and none of the ovarian carcinomas. One alteration found in the breast cancers was a 2-basepair (bp) deletion (4710delAG) which was subsequently shown to be a germline mutation, the other was a somatic missense mutation (Asp3095Glu) of unknown significance. Our results suggest that BRCA2 is a very infrequent target for somatic inactivation in breast and ovarian carcinomas, similar to the results obtained for BRCA1.

Immunogenetic Management Software: a new tool for visualization and analysis of complex immunogenetic datasets.

Here we describe the Immunogenetic Management Software (IMS) system, a novel web-based application that permits multiplexed analysis of complex immunogenetic traits that are necessary for the accurate planning and execution of experiments involving large animal models, including nonhuman primates. IMS is capable of housing complex pedigree relationships, microsatellite-based MHC typing data, as well as MHC pyrosequencing expression analysis of class I alleles. It includes a novel, automated MHC haplotype naming algorithm and has accomplished an innovative visualization protocol that allows users to view multiple familial and MHC haplotype relationships through a single, interactive graphical interface. Detailed DNA and RNA-based data can also be queried and analyzed in a highly accessible fashion, and flexible search capabilities allow experimental choices to be made based on multiple, individualized and expandable immunogenetic factors. This web application is implemented in Java, MySQL, Tomcat, and Apache, with supported browsers including Internet Explorer and Firefox on Windows and Safari on Mac OS. The software is freely available for distribution to noncommercial users by contacting Leslie.kean@emory.edu. A demonstration site for the software is available at http://typing.emory.edu/typing_demo , user name: imsdemo7@gmail.com and password: imsdemo.

Acute molecular response of mouse hindlimb muscles to chronic stimulation.

Stimulation of the mouse hindlimb via the sciatic nerve was performed for a 4-h period to investigate acute muscle gene activation in a model of muscle phenotype conversion. Initial force production (1.6 +/- 0.1 g/g body wt) declined 45% within 10 min and was maintained for the remainder of the experiment. Force returned to initial levels upon study completion. An immediate-early growth response was present in the extensor digitorum longus (EDL) muscle (FOS, JUN, activating transcription factor 3, and musculoaponeurotic fibrosarcoma oncogene) with a similar but attenuated pattern in the soleus muscle. Transcript profiles showed decreased fast fiber-specific mRNA (myosin heavy chains 2A and 2B, fast troponins T(3) and I, alpha-tropomyosin, muscle creatine kinase, and parvalbumin) and increased slow transcripts (myosin heavy chain-1beta/slow, troponin C slow, and tropomyosin 3y) in the EDL versus soleus muscles. Histological analysis of the EDL revealed glycogen depletion without inflammatory cell infiltration in stimulated versus control muscles, whereas ultrastructural analysis showed no evidence of myofiber damage after stimulation. Multiple fiber type-specific transcription factors (tea domain family member 1, nuclear factor of activated T cells 1, peroxisome proliferator-activated receptor-gamma coactivator-1alpha and -beta, circadian locomotor output cycles kaput, and hypoxia-inducible factor-1alpha) increased in the EDL along with transcription factors characteristic of embryogenesis (Kruppel-like factor 4; SRY box containing 17; transcription factor 15; PBX/knotted 1 homeobox 1; and embryonic lethal, abnormal vision). No established in vivo satellite cell markers or genes activated in our parallel experiments of satellite cell proliferation in vitro (cyclins A(2), B(2), C, and E(1) and MyoD) were differentially increased in the stimulated muscles. These results indicated that the molecular onset of fast to slow phenotype conversion occurred in the EDL within 4 h of stimulation without injury or satellite cell recruitment. This conversion was associated with the expression of phenotype-specific transcription factors from resident fiber myonuclei, including the activation of nascent developmental transcriptional programs.

Assignment of a locus for familial melanoma, MLM, to chromosome 9p13-p22.

Linkage analysis of ten Utah kindreds and one Texas kindred with multiple cases of cutaneous malignant melanoma (CMM) provided evidence that a locus for familial melanoma susceptibility is in the chromosomal region 9p13-p22. The genetic markers analyzed reside in a candidate region on chromosome 9p21, previously implicated by the presence of homozygous deletions in melanoma tumors and by the presence of a germline deletion in an individual with eight independent melanomas. Multipoint linkage analysis was performed between the familial melanoma susceptibility locus (MLM) and two short tandem repeat markers, D9S126 and the interferon-alpha (IFNA) gene, which reside in the region of somatic loss in melanoma tumors. An analysis incorporating a partially penetrant dominant melanoma susceptibility locus places MLM near IFNA and D9S126 with a maximum location score of 12.71. Therefore, the region frequently deleted in melanoma tumors on 9p21 presumably contains a locus that plays a critical role in predisposition to familial melanoma.

Resources for genetic management and genomics research on non-human primates at the National Primate Research Centers (NPRCs).

The National Primate Research Centers (NPRCs) established Working Groups (WGs) for developing resources and mechanisms to facilitate collaborations among non-human primate (NHP) researchers. Here we report the progress of the Genome Banking and the Genetics and Genomics WGs in developing resources to advance the exchange, analysis and comparison of NHP genetic and genomic data across the NPRCs. The Genome Banking WG has established a National NHP DNA bank comprising 1250 DNA samples from unrelated animals and family trios from the 10 NHP species housed within the NPRC system. The Genetics and Genomics WG is developing SNP arrays that will provide a uniform, highly informative, efficient and low-cost method for rhesus and long-tailed macaque genotyping across the eight NPRCs. This WG is also establishing a Biomedical Informatics Research Network-based portal for shared bioinformatics resources including vital statistics, genotype and population data and information on the National NHP DNA bank.

Skeletal myoblast transplantation for repair of myocardial necrosis.

Myocardial infarcts heal by scarring because myocardium cannot regenerate. To determine if skeletal myoblasts could establish new contractile tissue, hearts of adult inbred rats were injured by freeze-thaw, and 3-4.5 x 10(6) neonatal skeletal muscle cells were transplanted immediately thereafter. At 1 d the graft cells were proliferating and did not express myosin heavy chain (MHC). By 3 d, multinucleated myotubes were present which expressed both embryonic and fast fiber MHCs. At 2 wk, electron microscopy demonstrated possible satellite stem cells. By 7 wk the grafts began expressing beta-MHC, a hallmark of the slow fiber phenotype; coexpression of embryonic, fast, and beta-MHC continued through 3 mo. Transplanting myoblasts 1 wk after injury yielded comparable results, except that grafts expressed beta-MHC sooner (by 2 wk). Grafts never expressed cardiac-specific MHC-alpha. Wounds containing 2-wk-old myoblast grafts contracted when stimulated ex vivo, and high frequency stimulation induced tetanus. Furthermore, the grafts could perform a cardiac-like duty cycle, alternating tetanus and relaxation, for at least 6 min. Thus, skeletal myoblasts can establish new muscle tissue when grafted into injured hearts, and this muscle can contract when stimulated electrically. Because the grafts convert to fatigue-resistant, slow twitch fibers, this new muscle may be suited to a cardiac work load.

Spectrum of mutation and frequency of allelic deletion of the p53 gene in ovarian cancer.

BACKGROUND: The p53 gene encodes a nuclear phosphoprotein present in low levels in normal human cells. The wild-type form of this protein functions to restrain inappropriate cellular proliferation. Approximately one half of human epithelial ovarian cancers have mutations in the p53 gene and overexpress the mutant protein product. Deletion of one allele of the p53 gene also frequently occurs in these cancers. PURPOSE: We sought to define the spectrum of mutations in the p53 gene in epithelial ovarian cancer with respect to both the specific codons involved and the type of mutations observed. We also examined the frequency of allelic deletion of the p53 gene in cancers containing p53 gene mutations. METHODS: Tissue samples from the epithelial ovarian cancers of 62 patients were obtained during initial laparotomy. Histologic examination was done to ensure that the experimental samples used in this study contained more than 75% cancer cells. Total RNA was extracted from these samples and separately from matched control noncancerous regions of the surgical specimen or white blood cells. The purified RNAs were reverse transcribed to generate cDNA copies of exons 4-10 of the p53 gene. Two rounds of polymerase chain reaction (PCR) were conducted to produce enough template for DNA sequence analysis of the regions of interest within the p53 gene. Dideoxy sequencing of at least two independent productions of each amplified DNA template was done to confirm the validity of the mutations found. Allelic deletions were identified by PCR and gel electrophoretic techniques to examine three polymorphisms within the p53 gene in cancer-normal DNA pairs. RESULTS: We identified 45 mutations in exons 5-8 of the p53 gene, where mutations frequently have been found in other cancer types. An additional mutation was identified in exon 4. Overall, 72% of the mutations were transitions, 24% transversions, and 4% microdeletions. Allelic deletion of the other p53 allele was seen in 67% of ovarian cancers in which a p53 mutation was present. Germ-line p53 mutations were not found in any patients whose cancers had p53 mutations. CONCLUSIONS AND IMPLICATIONS: Like p53 mutations in other types of human cancers, those in epithelial ovarian cancers are diverse and occur frequently in exons 5-8. The predominance of transition mutations suggests that p53 mutations in ovarian cancer arise because of spontaneous errors in DNA synthesis and repair rather than the direct interaction of carcinogens with DNA. These molecular data are consistent with data from epidemiologic studies that have failed to demonstrate a convincing relationship between exposure to environmental carcinogens and the development of ovarian cancer.

Clonal origin of epithelial ovarian carcinoma: analysis by loss of heterozygosity, p53 mutation, and X-chromosome inactivation.

BACKGROUND: It has been suggested that multiple sites of epithelial ovarian carcinoma on the peritoneal surface reflect polyclonal disease arising from multiple primary tumors in the peritoneal mesothelium, rather than monoclonal disease spread by metastases from one primary ovarian cancer. PURPOSE: The purpose of this study was to investigate whether ovarian cancer has a monoclonal or polyclonal origin. METHODS: DNA specimens were obtained from peripheral blood lymphocytes (normal DNA) and from multiple tumor deposits of 17 women with epithelial ovarian carcinoma: primary tumors, metastatic deposits, and ascites. The clonal origin of each tumor was determined by performing (a) analysis to detect loss of heterozygosity at five loci on chromosomes 5, 11, 13, and 17; (b) sequencing of exons 5-8 of the p53 gene; and (c) X-chromosome inactivation analysis of the phosphoglycerate kinase (PGK) gene. RESULTS: In 15 of the 17 cases analyzed, there was clear evidence of monoclonal origin. The probability that the genetic events documented in these 15 cases occurred as independent events in each tumor deposit ranged from 2.5 x 10(-1) to 3.7 x 10(-16). In two cases, the pattern of allelic deletion and p53 gene mutation was compatible with either a monoclonal origin or origin from two primary ovarian tumors. CONCLUSIONS: The results did not support the hypothesis that ovarian cancer is a multifocal, polyclonal disease. Instead, the data suggest that sporadic epithelial ovarian carcinoma has either a monoclonal or a dual primary origin. IMPLICATIONS: These findings have important implications for understanding of the natural history of ovarian cancer and for clinical strategies aimed at prevention and early detection. Further studies will be required to determine the clonal origin of familial hereditary ovarian cancer.

Deletion mapping of a putative tumor suppressor gene on chromosome 4 in mouse lung tumors.

Genetic and molecular studies have implicated the region of the alpha-interferon gene cluster on mouse chromosome 4 as the location of a putative tumor suppressor gene. A region of homology on human chromosome 9p21-22 that is frequently deleted in multiple human cancers has recently been found to contain a candidate tumor suppressor gene called multiple tumor suppressor-1 (MTS1); which was previously shown to encode an inhibitor of cyclin-dependent kinase 4. We performed loss of heterozygosity and deletion analyses to map the most commonly deleted region on chromosome 4 in F1 hybrid mouse lung tumors. Ten simple sequence length polymorphism markers were analyzed with focus on the alpha-interferon region. Allelic losses were detected in 29 of 61 (48%) of the lung adenocarcinomas but in only 1 of 38 (3%) of the lung adenomas examined. In most cases, the losses appeared to occur by nondisjunction. However, in three carcinomas, we detected homozygous deletions that overlapped at simple sequence length polymorphism marker D4MIT77. These data suggest a critical region of about 2 cM immediately distal to the alpha-interferon locus as the likely domain of a novel tumor suppressor gene on mouse chromosome 4, the loss of which appears to be involved in the progression of mouse lung tumorigenesis.

Structure-activity studies of the hepatocarcinogenicities of alkenylbenzene derivatives related to estragole and safrole on administration to preweanling male C57BL/6J x C3H/HeJ F1 mice.

Further information on the structure-activity relationships among the synthetic and naturally occurring alkenylbenzene derivatives was obtained by examining their hepatocarcinogenicities for mice following administration of one or a few doses prior to weaning. Under these conditions preweanling male C3H/HeJ mice were more susceptible than male C57BL/6J mice or females of either strain to liver tumor induction by 1'-hydroxyestragole (1'-hydroxy-1-allyl-4-methoxybenzene) and 1'-hydroxysafrole (1'-hydroxy-1-allyl-3,4-methylenedioxybenzene). Male C57BL/6J X C3H/HeJ F1 mice given a single dose of 1'-hydroxyestragole at 12 days of age developed approximately twice as many hepatomas per liver as did those given the same dose per g of body weight at 1 day of age. The acetylenic compounds 1'-hydroxy-2',3'-dehydroestragole and 1'-hydroxy-2',3'-dehydrosafrole were the most potent derivatives studied; they were 5- and 10-fold more potent (based on the average numbers of hepatomas per liver) than the corresponding allylic benzene derivatives. 1'-Acetoxyestragole and 1'-acetoxysafrole had activities similar to those of their respective 1'-hydroxy derivatives; estragole derivatives were consistently 2- to 3-fold more potent than the related safrole derivatives. 1'-Hydroxyelemicin (1'-hydroxy-1-allyl-3,4,5-trimethoxybenzene), its acetic acid ester 1'-oxoestragole, and 3'-bromo-trans-anethole (3'-bromo-1-trans-propenyl-4-methoxybenzene) each had very weak, but statistically significant, hepatocarcinogenic activity. The propenylic derivatives cis-anethole, trans-isosafrole, 1:1 cis,trans-isosafrole, 3'-hydroxy-trans-anethole, piperine, and trans-cinnamaldehyde showed no hepatocarcinogenic activity at the levels examined. In contrast, the propenylic derivatives cis- and trans-asarone (1-propenyl-2,4,5-trimethoxybenzene) were each active; the hepatocarcinogenicities of the asarones were not inhibited by prior administration of pentachlorophenol, a sulfotransferase inhibitor that abolished the hepatocarcinogenicity of estragole under the same conditions. Furthermore, precocene II (6,7-dimethoxy-2,2-dimethyl-2H-1-benzopyran), a cyclic propenylic plant metabolite and asarone analogue, showed strong hepatocarcinogenic activity similar to that of 1'-hydroxy-2',3'-dehydroestragole and 1'-hydroxy-2',3'-dehydrosafrole; precocene I (the 7-methoxy analogue of precocene II) was less active than precocene II but more active than cis-asarone.

Genetic alterations of p53 and ras genes in 1,3-butadiene- and 2',3'-dideoxycytidine-induced lymphomas.

Mutations of p53 and ras genes were analyzed in 40 and 31 1,3-butadiene (BD)-induced lymphomas of B6C3F1 mice (BLFs), respectively, and in 63 2',3'-dideoxycytidine-induced lymphomas, which were collected from B6C3F1 (n = 16) or NIH Swiss mice (DLSs; n = 47). The frequencies of K- and N-ras mutations in BLFs (32 and 13%, respectively) were higher than those in DLSs (13 and 2%, respectively). Seven of 10 K-ras-mutated BLFs contained codon 13 CGC mutations, whereas no mutation in K-ras codon 13 was detected in DLSs, suggesting that the codon 13 CGC mutation is specific for BD exposure. Interestingly, 8 of 13 BLFs with ras mutations were from low-dose (< or = 200 ppm) or stop-exposure (26 weeks) groups. These results suggest that ras mutations play an important role in the development of BD-induced lymphoma and may represent an early event. Analysis of genetic alterations in exons 5-8 of the p53 gene revealed mutations in seven of the BLFs and three of the DLSs. All seven BLFs carrying p53 mutations were collected from the high-dose (625 ppm) continuous exposure group, which might indicate that p53 is involved in the progression of BD-induced lymphoma and in late stage of lymphomagenesis. Mutations in ras and p53 genes are relatively infrequent in 2',3'-dideoxycytidine-induced lymphomas, suggesting that other genes must be involved.

Major role of hepatic sulfotransferase activity in the metabolic activation, DNA adduct formation, and carcinogenicity of 1'-hydroxy-2',3'-dehydroestragole in infant male C57BL/6J x C3H/HeJ F1 mice.

1'-Hydroxy-2',3'-dehydroestragole is a synthetic acetylenic analogue of 1'-hydroxyestragole, the proximate carcinogenic metabolite of the naturally occurring hepatocarcinogen estragole (1-allyl-4-methoxybenzene). This analogue is considerably more potent than 1'-hydroxyestragole as an hepatocarcinogen in mice. 1'-Acetoxy-2',3'-dehydroestragole reacted readily with deoxyguanosine or deoxyguanosine 5'-monophosphate at neutrality to form two adducts. Adduct I, isolated and characterized after dephosphorylation of the deoxyguanosine 5'-monophosphate product, was a 1:1 mixture of two diastereomers of N2-(2',3'-dehydroestragol-1'-yl)deoxyguanosine. Adduct II was shown to be N-7-(2',3'-dehydroestragol-1'-yl)guanine. The reaction of deoxyadenosine with 1'-acetoxy-2',3'-dehydroestragole at neutrality produced Adducts III and IV. Adduct IV was characterized as N6-(2',3'-dehydroestragol-1'-yl)deoxyadenosine. Administration of [1'-3H]-1'-hydroxy-2',3'-dehydroestragole to male preweanling C57BL/6J x C3H/HeJ F1 (hereafter called B6C3F1) mice resulted in extensive covalent binding to hepatic DNA, RNA, and protein. On hydrolysis of the DNA to nucleosides, a single major adduct accounted for greater than 85% of the DNA-bound 3H. This adduct comigrated with Adduct I in two high performance liquid chromatography systems, had a pH partition profile identical to that of Adduct I, and was present as a mixture of diastereomers in a ratio of 2:1. The identity of the DNA adduct formed in vivo with Adduct I from the reaction of 1'-acetoxydehydroestragole indicated that a reactive ester was a major metabolic precursor in vivo. There was no significant loss of Adduct I from the hepatic DNA by 21 days after a single injection of a carcinogenic dose of 1'-hydroxy-2',3'-dehydroestragole. Adducts II, III, and IV were not detected in significant amounts in the hepatic DNA isolated by a phenol extraction method or by a more rapid hydroxylapatite method. Cytosolic sulfotransferase activity was demonstrated for 1-hydroxy-2',3'-dehydroestragole in mouse liver, and inhibition of this activity by greater than 95% was found on addition of 10 microM pentachlorophenol. The administration of pentachlorophenol (0.04 mumol/g body weight) 45 min prior to a single dose of 1'-hydroxy-2',3'-dehydroestragole (0.04 mumol/g body weight) in 12-day-old male B6C3F1 mice greatly inhibited (87-97%) the covalent binding of 1'-hydroxy-2',3'-dehydroestragole to hepatic macromolecules and the formation of hepatomas at 10 months.(ABSTRACT TRUNCATED AT 400 WORDS)

Further characterization of the DNA adducts formed by electrophilic esters of the hepatocarcinogens 1'-hydroxysafrole and 1'-hydroxyestragole in vitro and in mouse liver in vivo, including new adducts at C-8 and N-7 of guanine residues.

The identities of the adducts formed on reaction of the model electrophilic and carcinogenic esters 1'-acetoxysafrole or 1'-acetoxyestragole with deoxyguanosine in vitro and those formed in vivo in the hepatic DNA of 12-day-old male C57BL/6 X C3H/He F1 (hereafter called B6C3F1) mice treated with 1'-hydroxysafrole or 1'-acetoxysafrole were investigated further with more discriminating high-performance liquid chromatography systems than previously used. The adducts formed from the reactions of 1'-acetoxysafrole or 1'-acetoxyestragole are strictly analogous and are distinguished by the prefixes S and E, respectively. Five adducts, including S(E)-II identified by Phillips et al. (Cancer Res., 41: 176-186, 2664-2671, 1981) as N2-(trans-isosafrol-3'-yl)deoxyguanosine and the analogous isoestragole derivative, have been characterized from the reactions with each ester. Adducts S-I and E-I, tentatively identified by Phillips et al. as N2-(safrol-1'-yl)- or N2-(estragol-1'-yl)deoxyguanosine, were each resolved into a pair of diastereomers. The proposed structures for each diastereomer were confirmed by nuclear magnetic resonance and circular dichroism spectroscopy. Two new adducts, i.e., S(E)-V and S(E)-VI, were isolated from each reaction mixture. On the basis of their pKas, their loss of 3H from [8-3H]deoxyguanosine, their retention of 3H from [1',2'-3H]deoxyguanosine, and their nuclear magnetic resonance spectra, Adducts S-V and E-V were characterized as 8-(trans-isosafrol-3'-yl)- and 8-(trans-isoestragol-3'-yl)deoxyguanosine, respectively. Adducts S-VI and E-VI were characterized in a similar manner as 7-(trans-isosafrol-3'-yl)- and 7-(trans-isoestragol-3'-yl)guanine, respectively. Adducts S-III and E-III, minor components described in the earlier studies, were not observed in the present work. High-performance liquid chromatography of hydrolysates of the hepatic DNA of male 12-day-old B6C3F1 mice killed 9 h after a single dose (0.1 mumol/g body weight) of [2',3'-3H]-1'-hydroxysafrole showed that Adducts S-Ia, S-Ib, S-II, S-IV (identified by Phillips et al. as N6-(trans-isosafrol-3'-yl)deoxyadenosine), S-V, and S-VI were present at average levels of 3.5, 7.0, 24.4, 2.9, 1.2, and 3.6 pmol/mg DNA, respectively. Similar levels of these adducts were found in the hepatic DNA after administration of the same dose of [2',3'-3H]-1'-acetoxysafrole under identical conditions.

Allelotype analysis of 2',3'-dideoxycytidine- and 1,3-butadiene-induced lymphomas in B6C3F1 mice.

To identify potential tumor suppressor genes involved in lymphoma development, we generated allelotypes of 16 2',3'-dideoxycytidine (ddC and 31 1,3-butadiene (BD)-induced lymphomas from C57BL/6 x C3H/He F1 (hereafter called B6C3F1) mice. Two or more anonymous simple sequence length polymorphisms per autosome were examined for loss of heterozygosity (LOH). Allelic losses throughout the genome were generally infrequent, except for markers on chromosome 2, 4, 11 and 12. The highest frequency of allelic losses was observed on chromosome 12, with 38 and 39% in ddC and BD-induced lymphomas, respectively. The most prevalent LOH was localized to the distal region bounded by markers D12Mit263 and D12Nds2. No known tumor suppressor genes have been mapped to this region, and no obvious candidates could be identified, suggesting the presence of novel suppressor gene(s). LOH on chromosome 2 was observed in 31% of ddC-induced lymphomas but in only 3% (1/31) of BD-induced lymphomas, suggesting a ddC-specific genetic effect. Detailed analysis localized a potential tumor suppressor gene residing on the distal region of chromosome 2, between markers D2Mit147 and D2Mit148. Twenty-five % of ddC-induced and 23% of BD-induced lymphomas showed LOH on chromosome 4, and two discrete regions were identified. One of the regions includes the IFN gene cluster and is syntenic to human chromosome 9p2l-22. Candidate tumor suppressor genes, Mts1 (multiple tumor suppressor 1) and Mts2 have been mapped to this region. The second region is located on the distal part of chromosome 4, which is homologous to human chromosome 1p35-36, a region that is frequently deleted in various types of human tumors. Finally, 19% of ddC-induced and 29% of BD-induced lymphomas revealed LOH on chromosome 11 at the Acrb locus, which lies within 1 cM of p53, suggesting that the p53 tumor suppressor gene also plays a role in lymphomagenesis. These results suggest that multiple potential suppressor loci contribute to lymphoma development in B6C3F1 mice.

A deletion unit on chromosome 17q in epithelial ovarian tumors distal to the familial breast/ovarian cancer locus.

Linkage analysis in familial breast and ovarian cancer and studies of allelic deletion in sporadic ovarian tumors have suggested that chromosome 17q may be the location of a gene of importance in ovarian carcinogenesis. We have examined tumor and normal DNA samples from 120 patients with ovarian tumors for allelic deletion at 12 loci on chromosome 17q. Allelic deletion was observed in 64 cases (53%) of which 56 showed loss of heterozygosity at all loci analyzed on 17q. The pattern of allele loss at metastatic sites was consistent with loss of heterozygosity having occurred prior to metastasis. A common region of deletion, defined by 6 cases of invasive epithelial ovarian cancer and a benign serous cystadenoma, spanned 16 cM and was delimited by nm23 and GH. This region is distal to the region on chromosome 17q to which the familial breast/ovarian cancer susceptibility gene has been mapped. The results suggest that a tumor suppressor gene involved in sporadic ovarian carcinogenesis is located on the distal portion of chromosome 17q and is distinct from the gene linked to familial cases.

Detection of frequent allelic loss on proximal chromosome 17q in sporadic breast carcinoma using microsatellite length polymorphisms.

Analyses of losses of heterozygosity and linkage studies have implicated a gene(s) on chromosome 17q in the genesis of sporadic and early-onset familial breast carcinomas, respectively. To define the critical region of 17q, we examined DNAs from a series of 20 sporadic breast carcinomas and corresponding blood samples for allelic losses of chromosome 17q using microsatellite length polymorphisms. With these highly informative markers (average heterozygosity, 0.73), we observed frequent deletions of 17q at several loci. We found that D17S250 was deleted in 50% (7 of 14), THRA1 in 79% (11 of 14), D17S579 in 59% (11 of 19), NME1 in 29% (5 of 17), MPO in 36% (4 of 11), and GH in 25% (4 of 16) in the tumor set examined. A common region of deletion was found that was flanked by D17S250 to D15S579. These markers have recently been localized to a 6-cM interval of proximal chromosome 17q in bands 17q11.2-q21 and map within the region of the early-onset familial breast cancer locus, implying that the same gene or genes may be involved in both sporadic and familial breast tumors. Thyroid hormone receptor alpha and retinoic acid receptor alpha are two potential candidate genes in this region.