Biomedical subjects

M Wang

Publications and source records attributed to M Wang.

At least 667 records · Page 37Linked to original sources

PubMed1996

Brca2 is coordinately regulated with Brca1 during proliferation and differentiation in mammary epithelial cells.

We have analyzed the expression of the breast cancer susceptibility gene, Brca2, in mammary epithelial cells as a function of proliferation and differentiation. Our results demonstrate that Brca2 mRNA expression is tightly regulated during mammary epithelial proliferation and differentiation, and that this regulation occurs coordinately with Brca1. Specifically, Brca2 mRNA expression is up-regulated in rapidly proliferating cells; is down-regulated in response to serum deprivation; is expressed in a cell cycle-dependent manner, peaking at the G1/S boundary; and is up-regulated in differentiating mammary epithelial cells in response to glucocorticoids. In each case, an identical pattern of expression was observed for Brca1. These results indicate that proliferative stimuli modulate the mRNA expression of these two breast cancer susceptibility genes. In addition, the coordinate regulation of Brca1 and Brca2 revealed by these experiments suggests that these genes are induced by, and may function in, overlapping regulatory pathways involved in the control of cell proliferation and differentiation.

Animals↗

PubMed1996

Altered G1 checkpoint control determines adaptive survival responses to ionizing radiation.

Adaptive survival responses (ASRs) are observed when cells become more resistant to a high dose of a cytotoxic agent after repeated low dose exposures to that agent or another genotoxic agent. Confluent (G0/G1) human normal (GM2936B, GM2937A, AG2603, IMR-90), cancer-prone (XPV2359), and neoplastic (U1-Mel, HEp-2, HTB-152) cells were primed with repeated low doses of X-rays (ranging from 0.05-10 cGy/day for 4 days), then challenged with a high dose (290-450 cGy) on day 5. U1-Mel and HEp-2 cells showed greater than 2-fold transient survival enhancement when primed with 1-10 cGy. ASRs in U1-Mel or HEp-2 cells were blocked by cycloheximide or actinomycin D. Increases in cyclins A and D1 mRNAs were noted in primed compared to unirradiated U1-Mel and HEp-2 cells; however, only cyclin A protein levels increased. Cyclin D1 and proliferating cell nuclear antigen (PCNA) protein levels were constitutively elevated in HEp-2 and U1-Mel cells, compared to the other human normal and neoplastic cells examined, and were not altered by low or high doses of radiation. Low dose primed U1-Mel cells entered S-phase 4-6 h faster than unprimed U1-Mel cells upon low-density replating. Similar responses in terms of survival recovery, transcript and protein induction, and altered cell cycle regulation were not observed in the other human normal, cancer-prone or neoplastic cells examined. We hypothesize that only certain human cells can adapt to ionizing radiation by progressing to a point later in G1 (the A point) where DNA repair processes and radioresistance can be induced. ASRs in human cells correlated well with constitutively elevated levels of PCNA and cyclin D1, as well as inducibility of cyclin A. We propose that a protein complex composed of cyclin D1, PCNA, and possibly cyclin A may play a role in cell cycle regulation and DNA repair, which determine ASRs in human cells.

Adaptation, Physiological↗

PubMed1996

Induction of transplantation tolerance by chimeric donor/recipient class I RT1.Aa molecules.

Donor-specific transplantation tolerance was induced by administration of chimeric antigens in which four donor immunogenic amino acids (a.a.) were substituted onto the host class I MHC protein. We constructed chimeric rat RT1.Aa cDNA molecules by substituting nucleotides in the alpha1 helical region that encode 10 Lewis (LEW; RT1.A1) a.a., namely Asp58, Arg62, Glu63, Gln65, Lys66, Gly69, Asn70, Asn73, Ser77, and Asn80 ([alpha(1h)1]-RT1.Aa). The chimeric [alpha(1h)1]-RT1.Aa cDNA sequence was verified before transfection into Buffalo (BUF; RT1b) hepatoma cells. Interestingly, the helical regions of LEW rats (alpha(1h)1) and Wistar Furth (WF; RT1u) rats (alpha(1h)u) share four a.a. (Arg62, Glu63, Gln65, and Gly69). Consequently, subcutaneous administration of [alpha(1)1]-RT1.Aa transfectants (20x10(6); day -7) immunized BUF rats to reject in rapid fashion either LEW heart allografts (mean survival time [MST] = 4.2+/-0.4 days vs. 5.6+/-0.5 days in controls; P<0.001) or WF heart allografts (MST=4.4+/-0.6 days vs. 6.0+/-0.0 days in controls; P<0.002). Subcutaneous immunization of ACI (RT1a) rats with [a(1)1]-RT1.Aa transfectants (bearing 10 LEW donor a.a.) accelerated the rejection of LEW hearts (MST=5.0+/-0.8 days vs. 8.2+/-0.4 days in controls; P<0.001). In contrast, the same [a(1)1]-RT1.Aa transfectants (bearing only four WF donor a.a.) injected subcutaneously into ACI rats modestly prolonged the survival of WF hearts to 14.0+/-10.3 days from 5.4+/-0.5 days in controls (P<0.001). Furthermore, ACI recipients were rendered tolerant to WF heart allografts by a single injection via the portal vein of soluble [a(1)1]-RT1.Aa (but not RT1.Aa, RT1.Au, or [a(2)1]-RT1.Aa) antigens in conjunction with brief oral gavage treatment with cyclosporine. Thus, selected donor immunogenic a.a. (Arg62, Glu63, Gln65, and Gly69) of class I MHC antigens become tolerogenic when flanked by host sequences.

Amino Acid Sequence↗

PubMed1996

Crystal structures of the wild type and the Glu376Gly/Thr255Glu mutant of human medium-chain acyl-CoA dehydrogenase: influence of the location of the catalytic base on substrate specificity.

Crystal structures of the wild type human medium-chain acyl-CoA dehydrogenase (MCADH) and a double mutant in which its active center base-arrangement has been altered to that of long chain acyl-CoA dehydrogenase (LCADH), Glu376Gly/Thr255Glu, have been determined by X-ray crystallography at 2.75 and 2.4 A resolution, respectively. The catalytic base responsible for the alpha-proton abstraction from the thioester substrate is Glu376 in MCADH, while that in LCADH is Glu255 (MCADH numbering), located over 100 residues away in its primary amino acid sequence. The structures of the mutant complexed with C8-, C12, and C14-CoA have also been determined. The human enzyme structure is essentially the same as that of the pig enzyme. The structure of the mutant is unchanged upon ligand binding except for the conformations of a few side chains in the active site cavity. The substrate with chain length longer than C12 binds to the enzyme in multiple conformations at its omega-end. Glu255 has two conformations, "active" and "resting" forms, with the latter apparently stabilized by forming a hydrogen bond with Glu99. Both the direction in which Glu255 approaches the C alpha atom of the substrate and the distance between the Glu255 carboxylate and the C alpha atom are different from those of Glu376; these factors are responsible for the intrinsic differences in the kinetic properties as well as the substrate specificity. Solvent accessible space at the "midsection" of the active site cavity, where the C alpha-C beta bond of the thioester substrate and the isoalloxazine ring of the FAD are located, is larger in the mutant than in the wild type enzyme, implying greater O2 accessibility in the mutant which might account for the higher oxygen reactivity.

Acyl Coenzyme A↗

PubMed1996

Radiation lethality enhancement with 9-aminocamptothecin: comparison to other topoisomerase I inhibitors.

PURPOSE: Preclinical studies have demonstrated differences in potency, solubility, and tumor specific activity among the camptothecin (CPT) analogues. 9-Aminocamptothecin (9-AC) has demonstrated greater potency in animal studies than other clinically available Topoisomerase I (Topo I) inhibitors. We sought to determine: (a) if 9-AC enhanced the lethal effects of ionizing radiation to a greater extent than other Topo I inhibitors; and (b) the biological and biochemical characteristics of the enhancement. METHODS AND MATERIALS: Quiescent radioresistant human melanoma (U1-Mel) cells were x-irradiated (1-7 Gy) and exposed to various concentrations of 9-AC (0.1-100 microM), either before (for 4 h), during, or after (for 4 h) irradiation. Survival was determined via colony forming assays and normalized to correct for drug cytotoxicity. The effect of 9-AC on radiation-related potential lethal damage repair PLDR) (PLDR) was also measured. A modification of the SDS-KCl assay was used to quantify DNA-Topo I complexes. RESULTS: Enhancement of radiation lethality was observed using confluent U1-Mel cells. The sensitizer enhancement ratio (SER) after a 4 h postirradiation exposure of 10 microM 9-AC was 2.5 at 10% survival. Toxicity from the drug alone was greater than topotecan (TPT), but less than CPT. The radiation synergy effect was: (a) dependent on drug concentration (> or = 2 microM); (b) dependent on timing, with enhancement present only when the drug was present at the time of, or shortly after, radiation; and (c) irreversible, with inhibition of PLDR. Exposure to 9-AC during or after irradiation substantially elevated the number of DNA-Topo I complexes (four- to tenfold) over control levels and correlated with enhanced loss of survival. CONCLUSION: 9-Aminocamptothecin enhanced radiation lethality in vitro at low drug concentrations with characteristics similar to other Topo I inhibitors. A greater SER, but greater lethality with the drug alone, was obtained in comparison to TPT. The clinical implications of these findings remain unexplored.

Antineoplastic Agents↗

PubMed1996

Concentration and timing dependence of lethality enhancement between topotecan, a topoisomerase I inhibitor, and ionizing radiation.

PURPOSE: Topotecan (TPT) is a water-soluble Topoisomerase I (Topo I) inhibitor with reported antineoplastic activity against a variety of solid tumors (including nonsmall cell lung, small cell lung, ovarian, breast, esophageal, and head and neck primaries) and leukemias. We sought to determine: (1) if TPT enhanced the lethal effects of ionizing radiation: and (b) the biological and biochemical characteristics of the enhancement. METHODS AND MATERIALS: Quiescent human radioresistant melanoma (U1-Mel) cells were x-irradiated (1-12 Gy) and exposed to various TPT concentrations (0.1-300 microM) either before (for 4 h), during, or after (for 4 h) radiation. Survival was determined via colony forming assays and normalized to correct for drug cytotoxicity. The effects of TPT on radiation-related potential lethal damage repair (PLDR) and sublethal damage repair (SLDR) were measured. A modification of the SDS-KCl assay was used to quantify DNA-Topo I complexes. RESULTS: Enhanced radiation lethality by TPT was observed using quiescent U1-Mel cells. The sensitizer enhancement ratio (SER) after a 4 h postirradiation exposure of 4 microM TPT was 1.6 at 10% survival. The effect was: (A) dependent on drug concentration, with lethality enhancement and minimal drug lethality alone in the 2-10 microM range for a 4 h posttreatment; (b) dependent on timing, with enhancement observed only when drug was present at the time of, or shortly after, radiation; and (c) irreversible, with inhibition of PLDR and SLDR. Exposure to TPT during or after radiation substantially elevated DNA-Topo I complexes (four- to tenfold) over control levels and complex formation correlated to some degree with loss of survival. CONCLUSIONS: TPT enhanced radiation lethality in vitro at low drug concentrations that are clinically feasible. The rationale and design of an ongoing Phase I trial which utilizes concurrent TPT and radiation is discussed.

Antineoplastic Agents↗

PubMed1996

DNA-binding properties of Arabidopsis MADS domain homeotic proteins APETALA1, APETALA3, PISTILLATA and AGAMOUS.

The MADS domain proteins APETALA1 (AP1), APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG) specify the identity of Arabidopsis floral organs. AP1 and AG homocomplexes and AP3-PI heterocomplexes bind to CArG-box sequences. The DNA-binding properties of these complexes were investigated. We find that AP1, AG and AP3-PI are all capable of recognizing the same DNA-binding sites, although with somewhat different affinities. In addition, the three complexes induce similar conformational changes on a CArG-box sequence. Phasing analysis reveals that the induced distortion is DNA bending, oriented toward the minor groove. The molecular dissection of AP1, AP3, PI and AG indicates that the boundaries of the dimerization domains of these proteins vary. The regions required to form a DNA-binding complex include, in addition to the MADS box, the entire L region (which follows the MADS box) and the first putative amphipathic helix of the K box in the case of AP3-PI, while for AP1 and AG only a part of the L region is needed. The similarity of the DNA-binding properties of AP1, AP3-PI and AG is discussed with regard to the biological specificity that these proteins exhibit.

AGAMOUS Protein, Arabidopsis↗

PubMed1996

In vitro induction of benzo(a)pyrene diol epoxide-DNA adducts in peripheral lymphocytes as a susceptibility marker for human lung cancer.

Given the same exposure, DNA adduct profiles can be considered as a phenotypic marker for carcinogen metabolism and DNA repair, which may reflect individual susceptibility to chemical carcinogenesis. Based on this notion, we have established a straightforward assay that measures induced DNA adducts in peripheral lymphocytes exposed in vitro to a model carcinogen, benzo(a)pyrene diol epoxide (BPDE) by 32P-postlabeling. To test the hypothesis that the levels of induced DNA adducts are a predictor for cancer risk, we conducted a pilot study of 21 lung cancer patients and 41 healthy frequency-matched controls. We found that the peripheral lymphocytes of cancer patients tended to accumulate higher levels of BPDE-DNA adducts than controls did (mean +/- SE of relative adduct labeling x 10(7) value; 59.6 +/- 12.0 versus 39.4 +/- 6.1 for cases and controls, respectively; P = 0.09). Using the tertile relative adduct labeling value of controls (10 adducts/10(7) nucleotides) as the cutoff point, 18 of 21 cases and 23 of 41 controls distributed above this level (odds ratio, 4.7; 95% confidence interval, 1.2-18.5). In logistic regression analysis, the level of induced adduct was an independent risk factor (odds ratio, 6.4; 95% confidence interval, 1.3-29.4) after adjustment for the potential confounding factors, i.e., age, sex, ethnicity, and smoking. Stratified analyses showed that greater differences in DNA adduct levels induced by BPDE between cases and controls were observed in individuals younger than 65 years and in nonsmokers. Despite the small sample size, the significant association between the level of BPDE-induced DNA adducts and risk for lung cancer suggests that this assay is a promising method for further investigations.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1996

Mevalonic acid is limiting for N-linked glycosylation and translocation of the insulin-like growth factor-1 receptor to the cell surface. Evidence for a new link between 3-hydroxy-3-methylglutaryl-coenzyme a reductase and cell growth.

Depletion of mevalonic acid (MVA), obtained by inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase using lovastatin, depressed the biosynthesis of dolichyl-phosphate and the rate of N-linked glycosylation and caused growth arrest in the melanoma cell line SK-MEL-2. The growth arrest was partially prevented by addition of high concentrations of insulin-like growth factor-1 (IGF-1) to the cells, indicating that MVA depletion may inhibit cell growth through decreasing the number of IGF-1 receptors (IGF-1R) at the cell surface. Such a decrease in receptor number might be a result of a lowered translocation of de novo synthesized receptors to the cell membrane which in turn might be a result of a decreased N-linked glycosylation of the receptor proteins. We could also demonstrate that IGF-1R became underglycosylated and that the amount of de novo synthesized IGF-1R proteins at the cell membrane was drastically decreased upon MVA depletion. Analysis of receptor proteins cross-linked with IGF-1, as well as binding assays and immunocytostaining confirmed that the number of functional membrane-bound IGF-1R was substantially reduced. The N-linked glycosylation and the expression of de novo synthesized IGF-1R proteins at the cell surface as well as the number of IGF-1 binding sites were completely restored upon replenishment of MVA. These effects of MVA were efficiently abrogated by the glycosylation inhibitor tunicamycin. The translocation of IGF-1R to the cell membrane was shown to take place just prior to initiation of DNA synthesis in arrested cells stimulated with MVA. Additionally, there was a clear correlation between IGF-1 binding and initiation of DNA synthesis with regard to the MVA dose requirement. It was confirmed that inhibition of HMG-CoA reductase activity and N-linked glycosylation also depressed the expression of functional IGF-1R in other cell types (i.e. hepatoblastoma cells and colon cancer cells). Our data suggest that this mechanism is involved in MVA-regulated cell growth.

Base Sequence↗

PubMed1996

Delineation of 7q11.2 deletions associated with Williams-Beuren syndrome and mapping of a repetitive sequence to within and to either side of the common deletion.

The majority of Williams-Beuren syndrome (WBS) patients have been shown to have a microdeletion within 7q11.2 including the elastin gene locus. The extent of these deletions has, however, not been well characterized. Thirty-five deletion patients were tested for all polymorphic markers in the 7q11.2 region bounding ELN to define the extent of deletions associated with WBS. With only one exception, ELN, D7S1870, and one copy of the D7S489 locus (D7S489U) were always included in the deletions. One patient showed lack of maternal inheritance at D7S1870 and not at ELN or D7S489U. A product corresponding to D7S489U was amplified from YAC 743G6 and from the P1 clone RMC07P008, thereby localizing both to within the common deletion. The boundary of the deleted region on the proximal (centromeric) side is D7S653 and on the distal side is D7S675, neither of which were ever included in the deletion. One locus, D7S489L, was variably deleted in patients, indicating a minimum of two common breakpoints on the proximal side. At least one additional repeat amplified by D7S489 (D7S489M) was localized to a YAC contig mapping distal to the common deletion. The D7S489 sequence is highly homologous to several cDNA clones in the GenBank database and contains an Alu sequence. It is possible that this andsolidusor other repetitive sequences in this region could play a role in the mechanism of deletion.

Base Sequence↗

PubMed1996

A simple method for detecting human polyomavirus DNA in urine by the polymerase chain reaction.

In order to develop a simple and sensitive method for detecting human polyomavirus DNA in the urine of patients by the polymerase chain reaction (PCR), it was found that the viral DNA could be released from urine by proteinase K and then amplified by PCR directly, without additional treatment such as ultracentrifugation or DNA extraction. Direct PCR amplification of viral DNA from urine was volume limited and 5 microliters of urine appeared to be the optimum amount for direct PCR amplification. When the urine volume was greater than 10 microliters, the results of PCR were inconsistent. However, the urine volume could be increased after dialysis to remove possible inhibitor(s) which may interfere with PCR. Direct PCR amplification of patient urine is convenient and eliminates several steps which can cause loss of DNA from the sample.

DNA, Viral↗

PubMed1996

Secretagogue-induced proteolysis of cAMP-dependent protein kinase in intact rat alveolar epithelial type II cells.

Stimulation of secretion from rat alveolar epithelial type II cells by the beta-adrenergic agonist terbutaline activates cAMP-dependent protein kinase (PKA). The same secretagogue also activates endogenous protease calpain in type II cells. In this study, we investigated the effect of calpain activation on PKA and its phosphorylation activity in stimulated type II cells. Type II cells were either pretreated with cell-permeable calpain specific inhibitor (N-acetyl-leucyl-leucyl-methioninal) or untreated, and subsequently stimulated with terbutaline. Stimulus-induced phosphorylation activity was assayed using the PKA-specific substrate Kemptide. Maximum PKA activity was observed within 1-3 min of stimulation. Peak activity of the untreated cells was 20-25% higher and longer than that of the inhibitor-treated cells. The stimulus-induced phosphorylation activity of both cell groups was suppressable by PKA-specific inhibitor. Concomitant photoaffinity labeling with radioactive 8-azido-cAMP revealed that a 39 kDa proteolytic fragment was generated in response to stimulation by terbutaline. Stimulus-induced activation of PKA resulted in the phosphorylation of two endogenous proteins, p112 and p47. Phosphorylation of p112 and p47 was modulated in cells pretreated with calpain inhibitor or in the presence of PKA inhibitor. Aggregate results indicate that stimulus-induced proteolysis of pKA occurs in type II cells, suggesting that limited proteolysis of PKA by endogenous calpain may convert an initial transient signal to sustained and augumented phosphorylation activity for secretion.

Adrenergic beta-Agonists↗

PubMed1996

Mutation spectra of M13 vectors containing site-specific Cis-Syn, Trans-Syn-I, (6-4), and Dewar pyrimidone photoproducts of thymidylyl-(3'-->5')-thymidine in Escherichia coli under SOS conditions.

The mutations spectra of cis-syn, trans-syn-I, (6-4), and Dewar pyrimidone photoproducts of the TT site of AATTAA and TATTAT in the (-) strand of a heteroduplex M13 vector were obtained in an excision and photoreversal repair deficient Escherichia coli host under SOS conditions. Oligonucleotides containing site-specific photoproducts were annealed to a complementary uracil-containing (+) strand that contained one or more unique pairs of nucleotide mismatches and used to prime (-) strand synthesis with a DNA polymerase and dNTPs. Following DNA synthesis, the reaction mixtures were incubated with T4 DNA ligase and ATP and then used to transfect SOS-induced competent CSRO6F' cells (uvrA6 and phr-1). The transfectants were plated, gridded, and probed by oligonucleotides specific for progeny of the (-) and (+) strands. Individual progeny of the photoproduct-containing (-) strands were plaque purified and sequenced by the dideoxy method. The cis-syn and trans-syn-I dimers were found not to be very mutagenic (<9%), the Dewar product more so (<33%), and the (6-4) product the most mutagenic (<73%). The mutation spectra were similar to those previously reported for the same photoproducts of the TT site of AGTTGG in the (+) strand of an M13 vector [Lawrence, C. W., et al. (1990) Mol. Gen Genet. 222, 166-168; LeClerc, J. E., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9685-9689] except that -1 deletion mutations were not observed for the trans-syn-I photoproducts, and a lower frequency of 3'-T-->C mutations was observed for the (6-4) photoproduct. Evidence that a small percentage of (+) strand repair of a double mismatch to the 3'-side of the photoproduct. Evidence that a small percentage of (+) strand repair of a double mismatch to the 3'-side was obtained from transfection experiments in which a second double mismatch was introduced opposite or flanking the photoproduct. Analysis of the minor tandem mutations induced by the (6-4) and Dewar products suggests that the SOS polymerase complex is able to elongate what amounts to double mismatches opposite these photoproducts and is consistent with the action of a highly processive polymerase that lacks proofreading ability.

Bacteriophage M13↗

PubMed1996

Induction of specific allograft immunity by soluble class I MHC heavy chain protein produced in a baculovirus expression system.

Spodoptera frugiperda (Sf9) insect cells secreted a class I MHC RT1.Aa heavy chain protein when infected with baculovirus that bore a construct that contained a honeybee melittin secretion (ms) signal attached to RT1.Aa cDNA. The RT1.Aa heavy chain protein in the culture supernatant and cell lysate immunoprecipitated in the presence of 5 individual anti-RT1.Aa-specific mAb. As was revealed by densitometric analysis, the ms signal increased the production (7- to 17-fold) and secretion (20- to 47-fold) of RT1.Aa protein by Sf9 cells (compared with RT1Aa-Sf9 cells without the ms signal). Subcutaneous immunization with secreted RT1.Aa heavy chain protein of Wistar-Furth (WF; RT1u) rats (day -4) accelerated the rejection of ACI (RT1a), but not third-party Brown Norway (BN; RT1n), heart allografts from 5.9 +/- 0.5 days in controls to 4.0 +/- 0.0 days (P < 0.001); cell lysate from RT1.Aa-Sf9 or ms/RT1.Aa-Sf9 cells reduced ACI heart allograft survival to 3.8 +/- 0.4 days or 3.7 +/- 0.5 days, respectively (P < 0.001). Indirect presentation of RT1.Aa heavy chain proteins by syngeneic macrophages shortened the survival of RT1.Aa-disparate PVG.R8 (RT1.AaDuBuCu) heart allografts in PVG.1U (RT1u) hosts from 6.3 +/- 0.5 days in controls to 4.0 +/- 0.0 days (P < 0.01). Finally, RT1.Aa heavy chain proteins injected into the thymus or into the portal vein (day -14) in combination with anti-T cell receptor mAb (days -14 and -13) induced indefinite survival of ACI liver allografts in Lewis (RT1l) recipients ( > 250 days). Thus, indirect presentation of soluble class I MHC heavy chain proteins (produced in a baculovirus/Sf9 cell system) may either sensitize or induce tolerance in the same fashion as native class I MHC alloantigens expressed on donor tissues.

Animals↗

PubMed1996

Beneficial effect of graft perfusion with anti-T cell receptor monoclonal antibodies on survival of small bowel allografts in rat recipients treated with brequinar alone or in combination with cyclosporine and sirolimus.

In the present experiments, a multimodality regimen was developed that included an anti-T cell receptor R73 monoclonal antibody and the pharmacologic agents brequinar (BQR), cyclosporine (CsA), and sirolimus (rapamycin; RAPA) to prolong the survival of small bowel (SB) allografts. BQR was the most potent single drug: the 4.0 or 8.0 mg/kg/day BQR doses delivered every second day (q.o.d.) per gavage for 28 days prolonged the survival of Brown Norway (BN; RT1n) SB allografts in Lewis (LEW; RT1l) recipients from a mean survival time of 10.6 +/- 1.9 days in untreated controls to 29.2 +/- 5.8 days, respectively (both P < 0.001). When treatment was extended to 56 days, 8.0 mg/kg/q.o.d BQR produced a mean survival time of 83.8 +/0 33.8 days (P < 0.001), with 2/5 hosts surviving more than 100 days. In a host-versus-graft model, BQR (8.0 mg/kg/q.o.d) delivered for 28 days with CsA (2.0 mg/kg/day) and RAPA (0.04 mg/kg/day) delivered intravenously for 14 days prolonged the survival of BN SB grafts in LEW recipients to 54.4 +/- 21.0 days (P < 0.001). Extending triple-drug therapy to 42 days induced the prolongation of SB allograft survival to greater than 100 days in 5/7 recipients. Although pretransplant perfusion of the grafts with R73 mAb was ineffective alone, the combination of graft perfusion and a 28-day course of BQR (8.0 mg/kg/q.o.d) in the GVH model indefinitely prolonged LEW graft in F1 recipients. Alternatively, indefinite survival of SB allografts ( > 100 days; P < 0.001) was achieved by the combination of a 14-day course of a triple-drug regimen using each agent at subtherapeutic doses, namely BQR (2.0 mg/kg/q.o.d.), CsA (2.0 mg/kg/day), and RAPA (0.04 mg/kg/day). The state of transplantation tolerance is these hosts was documented by the acceptance of donor-type but not third-party heart allografts.

Animals↗

PubMed1996

Aromatic DNA adducts in adjacent tissues of breast cancer patients: clues to breast cancer etiology.

The etiology of the majority of human breast cancers is unknown. Environmental factors have long been suspected to play a role, but no specific causative agent has been identified. If the hypothesis that environmental carcinogen exposure contributes to human breast cancer is true, carcinogen-DNA adducts would be expected to be present in human breast tissues. To address this possibility, aromatic DNA adducts were measured in 87 surgical specimens of normal human breast tissues from 87 breast cancer patients undergoing mastectomy using the nuclease P1-enhanced version of the 32P postlabeling assay. Breast tissue samples from 29 noncancer patients undergoing reduction mammoplasty served as controls. Whereas aromatic DNA adducts were detected in all tissue samples examined, the total adduct levels in cancer patients were significantly higher than that in noncancer controls [mean +/- SEM, 97.4 +/- 23.4/10(9) nucleotides (range, 3.8-1737.1) versus 18.1 +/- 11.6/10(9) nucleotides (range, 5.6-56.7), respectively; P < 0.01, t test and Mann-Whitney test]. This difference was not affected by the age distribution of the two groups. The typical smoking-related DNA adduct pattern (i.e., a diagonal radioactive zone) was observed in 29 of 87 tissues (17 of 17 current smokers, 5 of 8 former smokers, 4 of 52 nonsmokers, and 3 of 10 patients with unknown smoking status) and in 2 of 10 control tissues. It was of interest that a benzo(a)pyrene (BP)-like DNA adduct was observed in 36 normal adjacent breast tissues (41%), 27 of which were from nonsmokers. Levels of this BP-like adduct were extremely high (> 100/10(9) nucleotides) in 5 patients (4 nonsmokers and 1 smoker) and moderately high (> 10/10(9) nucleotides) in 13 other patients (8 nonsmokers and 5 smokers). One patient exhibited this adduct at a level of 1500/10(9) nucleotides, which is comparable to the highest level of total adducts reported in human tissues related to carcinogen exposure (e.g., cigarette smoking). In contrast, this adduct was absent (< 1/10(9) nucleotides) in all of the control tissues. Cochromatography and rechromatography analysis of DNA samples from human breast tissues and from MCF-7 cells treated with BP revealed that this adduct could be generated by BP exposure but is not the major BP 7,8-diol-9,10-epoxide-deoxyguanine adduct detected previously in animal tissues and human mammary epithelial cells. These findings support the hypothesis that environmental carcinogen exposure, in addition to cigarette smoking, may be associated with the etiology of human breast cancer.

Adult↗

PubMed1996

Therapeutic antitumor response after immunization with a recombinant adenovirus encoding a model tumor-associated antigen.

Recombinant adenovirus (rAd), deleted of critical genes that enable viral replication and replaced with genes encoding heterologous proteins, has been shown to be a safe and effective vector in gene therapy studies. To evaluate a potential role for rAd as an immunogen, we used two different replication-defective type 2 rAds encoding the model Ag, beta-galactosidase (beta-gal). To determine whether rAd elicited the kind of immune responses therapeutic in an anti-tumor setting, the beta-gal-expressing adenocarcinoma, CT26.CL25, was used. Splenocytes from BALB/c mice immunized with 1 x 10(7) infectious units (iu) of rAd demonstrated anti-beta-gal activity after in vitro culture with the relevant L(d) beta-gal peptide. Adoptive transfer of these same splenocytes produced dramatic regression of established pulmonary metastases. However, when tumor-bearing mice were treated with 1 x 10(7) iu of rAd, no reduction in established disease was observed even when rAd was given with exogenous IL-2. To increase the viral dose delivered to each animal, we used an E1-E4-deleted rAd that could be grown to much higher titers. Significant reduction occurred with 10-fold more rAd (1 x10(8) iu) was administered. Exogenous IL-2 administration with 1 x 10(8) iu of rAd resulted in augmentation of this anti-tumor effect. These findings demonstrate that when using a nonreplicating virus, the viral dose is directly related to the immune response generated. These data constitute the first reported use of rAd in the treatment of an established experimental cancer and may have implication for the treatment of human cancer.

Adenocarcinoma↗

PubMed1996

Gene therapy for head and neck cancer. Comparing the tumor suppressor gene p53 and a cell cycle regulator WAF1/CIP1 (p21).

OBJECTIVE: To compare the efficacy of the tumor suppressor gene wild-type p53 with that of cell-cycle regulator WAF1/CIP1 as single-agent gene therapy for squamous cell carcinoma of the head and neck. EXPERIMENTAL METHODS AND DESIGN: Recombinant cytomegalovirus-promoted adenoviruses containing the wild-type p53 or WAF1/CIP1 (p21) genes were transiently introduced into squamous cell carcinoma of the head and neck cell lines. Standard Western blot analysis was used to determine expression in these cells of the proteins encoded by these genes. A nude mouse xenograft model of squamous cell carcinoma of the head and neck was used to investigate the in vivo efficacy of the repeated gene therapy interventions. RESULTS: Western blot analysis showed marked induction of the WAF1/CIP1 tumor suppressor gene product by both the p21 adenovirus and the wild-type p53 adenovirus (as a secondarily transcribed product). In vitro growth curves demonstrated that the wild-type p53 adenovirus significantly inhibited cell growth in these cell lines, whereas direct induction of the p21 gene product did not. Repeated infection with wild-type p53 adenovirus significantly reduced the size of established subcutaneous tumors, whereas infection with a replication-defective viral control did not. CONCLUSION: Wild-type p53 adenovirus exhibits substantial in vitro and in vivo tumor suppressor activity in squamous cell carcinoma of the head and neck cell lines. This tumor suppression is not a function of the induced WAF1/CIP1 (p21) transcriptional product. Further studies are required to investigate the potential for induction of apoptosis by gene therapy.

Adenoviruses, Human↗