DEPLETION OF O-6-ALKYLGUANINE - DNA ALKYLTRANSFERASE BY O-6-BENZYLGUANINE IN 3-DIMENSIONAL COLLAGEN CULTURES OF NORMAL HUMAN BREAST EPITHELIAL-CELLS

被引:5
|
作者
HAMDAN, MAMS
GUZMAN, RC
YANG, J
BEATTIE, C
MASCHARACK, P
NANDI, S
机构
[1] USDA,CLAY CTR,NE 68933
[2] UNIV CALIF SANTA CRUZ,DEPT CHEM & BIOCHEM,SANTA CRUZ,CA 95064
关键词
D O I
10.1093/carcin/13.10.1743
中图分类号
R73 [肿瘤学];
学科分类号
100214 ;
摘要
O6-Alkylguanine-DNA alkyltransferase (AGT) is a protein which removes the promutagenic O6-alkylguanine lesion induced in DNA by alkylating agents. Our results demonstrate that freshly isolated organoids from reduction mammoplasty specimens contain significant levels of AGT activity. AGT activity in breast epithelial cells shows interindividual variation. Constitutive levels of AGT activity remain unchanged during short-term serum-free culture of breast epithelial cells inside three-dimensional rat-tail collagen gel matrix. In the present study, we optimized conditions for depleting AGT activity in human breast epithelial cells cultured in three-dimensional collagen gel matrix using O6-methylguanine and O6-benzylguanine which are substrates for AGT. AGT activity was efficiently inactivated by exposure of cells to O6-methylguanine or O6-benzylguanine. Inactivation with O6-benzylguanine was more rapid, of greater magnitude and consistency and occurred at lower concentrations than with O6-methylguanine. Near-complete inactivation (> 99.5%) of AGT activity was reproducibly achieved with 50 muM O6-benzylguanine. In contrast, 500 muM O6-methylguanine was needed to obtain a maximal effect and this reduced AGT activity by only 53-93% of control. Within 30 min of adding the free base, 50 muM O6-benzylguanine depleted 95% of the levels of AGT compared to 30% inhibition with 500 muM O6-methylguanine. The profile for restoration of AGT activity was different following a 24 h incubation and subsequent removal of each of the guanine derivatives. AGT activity levels remained undetectable for at least 2 days after removal of 50 muM O6-benzylguanine from the medium and recovered to only 53% of control values after an additional 3 days. AGT activity levels remained undetectable for at least 2 days after removal of 50 muM O6-benzylguanine from the medium and recovered to only 53% of control values after an additional 3 days. In contrast, following removal of 500 muM O6-methylguanine, the activity was restored from its nadir of 16% of control values reaching pretreatment levels after 5 days. These results suggest that treatment with O6-benzylguanine may be used to modulate the incidence of transforming mutations in cultured human breast epithelial cells treated with chemical carcinogens which give rise to O6-alkylguanine adducts.
引用
收藏
页码:1743 / 1749
页数:7
相关论文
共 50 条
  • [21] Role of codon 160 in the sensitivity of human O6-alkylguanine-DNA a alkyltransferase to O6-benzylguanine
    Xu-Welliver, M
    Leitao, J
    Kanugula, S
    Meehan, WJ
    Pegg, AE
    BIOCHEMICAL PHARMACOLOGY, 1999, 58 (08) : 1279 - 1285
  • [22] In vitro inactivation of human O-6-alkylguanine DNA alkyltransferase by antitumor triazene compounds
    Lacal, PM
    DAtri, S
    Orlando, L
    Bonmassar, E
    Graziani, G
    JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, 1996, 279 (01): : 416 - 422
  • [23] Repair of O-6-benzylguanine by the Escherichia coli Ada and Ogt and the human O6-alkylguanine-DNA alkyltransferases
    Goodtzova, K
    Kanugula, S
    Edara, S
    Pauly, GT
    Moschel, RC
    Pegg, AE
    JOURNAL OF BIOLOGICAL CHEMISTRY, 1997, 272 (13) : 8332 - 8339
  • [24] O6-benzylguanine suppression of O6-alkylguanine-DNA alkyltransferase in anaplastic gliomas
    Schold, SC
    Kokkinakis, DM
    Chang, SM
    Berger, MS
    Hess, KR
    Schiff, D
    Robins, HI
    Mehta, MP
    Fink, KL
    Davis, RL
    Prados, MD
    NEURO-ONCOLOGY, 2004, 6 (01) : 28 - 32
  • [25] Isolation of human O6-alkylguanine-DNA alkyltransferase mutants highly resistant to inactivation by O6-benzylguanine
    Xu-Welliver, M
    Kanugula, S
    Pegg, AE
    CANCER RESEARCH, 1998, 58 (09) : 1936 - 1945
  • [26] MUTATIONS IN THE ADA O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE CONFERRING SENSITIVITY TO INACTIVATION BY O-6-BENZYLGUANINE AND 2,4-DIAMINO-6-BENZYLOXY-5-NITROSOPYRIMIDINE
    CRONE, TM
    KANUGULA, S
    PEGG, AE
    CARCINOGENESIS, 1995, 16 (08) : 1687 - 1692
  • [27] DIFFERENTIAL INACTIVATION OF MAMMALIAN AND ESCHERICHIA-COLI O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASES BY O-6-BENZYLGUANINE
    ELDER, RH
    MARGISON, GP
    RAFFERTY, JA
    BIOCHEMICAL JOURNAL, 1994, 298 : 231 - 235
  • [28] O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE ACTIVITY IN NORMAL HUMAN-TISSUES AND CELLS
    GRAFSTROM, RC
    PEGG, AE
    TRUMP, BF
    HARRIS, CC
    CANCER RESEARCH, 1984, 44 (07) : 2855 - 2857
  • [29] ALTERATION OF ARGININE-128 TO ALANINE ABOLISHES THE ABILITY OF HUMAN O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE TO REPAIR METHYLATED DNA BUT HAS NO EFFECT ON ITS REACTION WITH O-6-BENZYLGUANINE
    KANUGULA, S
    GOODTZOVA, K
    EDARA, S
    PEGG, AE
    BIOCHEMISTRY, 1995, 34 (21) : 7113 - 7119
  • [30] O6-ALKYLGUANINE-DNA ALKYLTRANSFERASE ACTIVITY IN A HUMAN MEDULLOBLASTOMA CELL-LINE TREATED WITH O6-BENZYLGUANINE
    KOALA, D
    SILBER, J
    BERGER, M
    CLINICAL RESEARCH, 1993, 41 (01): : A73 - A73