Rossiello M R, Rao P M, Rajalakshmi S, Sarma D S
Department of Pathology, University of Toronto, Ontario, Canada.
Mol Carcinog. 1994 Aug;10(4):237-45. doi: 10.1002/mc.2940100408.
Our earlier studies demonstrated that the beta-hydroxy-beta-methylglutaryl coenzyme A (HMG CoA) reductase gene is hypomethylated and overexpressed in hepatic nodules initiated by 1,2-dimethylhydrazine (1,2-DMH). The study presented here examined whether the pattern of DNA methylation of the HMG CoA reductase gene in hepatic nodules reflected carcinogen-DNA interaction during initiation. Accordingly, hepatic nodules were generated in male Fischer 344 rats with either 1,2-DMH or aristolochic acid (AA), which interact predominantly with the guanine and adenine bases in DNA, respectively. DNA from individual nodules was restricted with HpaII, MspI, and HhaI, and the fragments obtained were hybridized to a cDNA probe for HMG CoA reductase. The results indicated that the hypomethylation pattern in the reductase gene in the nodules initiated with these two carcinogens was similar, although they interacted with different bases in the DNA. The question still remained whether the DNA fragments obtained by digesting the two sets of nodules with the restriction endonucleases were from the same domains in the genome of HMG CoA reductase. To examine this, probes for the different domains of the HMG CoA reductase gene were generated from the cDNA using the restriction enzyme Accl. Three probes were obtained: (i) a 5'-end fragment corresponding to the membrane-spanning region, (ii) a second fragment corresponding to the 3'-end of the protein, and (iii) a third fragment spanning the region between (i) and (ii). Each of these probes was radiolabeled and hybridized to the HpaII- and HhaI-generated fragments from the DNA of hepatic nodules initiated with 1,2-DMH and AA. Irrespective of the carcinogen used for initiation, hypomethylation occurred in all three domains of the gene. More important, the pattern of hypomethylation was similar in the nodules initiated by the two carcinogens. Furthermore, an overall similarity was seen in the hypomethylation patterns in the c-myc and c-Ha-ras genes in the DNA of nodules initiated with either 1,2-DMH or AA. These results raise the possibility that the pattern of hypomethylation established in the hepatic nodules may not directly reflect the interaction between the initiating carcinogen and DNA but may represent a unique phenotype of hepatic nodules.
我们早期的研究表明，β-羟基-β-甲基戊二酰辅酶A（HMG CoA）还原酶基因在由1,2-二甲基肼（1,2-DMH）引发的肝结节中发生低甲基化并过度表达。本文所呈现的研究探讨了肝结节中HMG CoA还原酶基因的DNA甲基化模式是否反映了启动过程中致癌物与DNA的相互作用。因此，在雄性Fischer 344大鼠中分别用1,2-DMH或马兜铃酸（AA）诱导产生肝结节，这两种物质分别主要与DNA中的鸟嘌呤和腺嘌呤碱基相互作用。从各个结节中提取的DNA用HpaII、MspI和HhaI进行酶切，得到的片段与HMG CoA还原酶的cDNA探针杂交。结果表明，尽管这两种致癌物与DNA中的不同碱基相互作用，但由它们引发的结节中还原酶基因的低甲基化模式相似。问题仍然存在，即通过用限制性内切酶消化这两组结节获得的DNA片段是否来自HMG CoA还原酶基因组中的相同区域。为了对此进行研究，使用限制性内切酶Accl从cDNA中制备了HMG CoA还原酶基因不同区域的探针。得到了三个探针：（i）一个对应于跨膜区域的5'端片段，（ii）一个对应于蛋白质3'端的第二个片段，以及（iii）一个跨越（i）和（ii）之间区域的第三个片段。将这些探针分别进行放射性标记，并与用1,2-DMH和AA引发的肝结节DNA经HpaII和HhaI酶切产生的片段杂交。无论用于启动的致癌物是什么，该基因的所有三个区域均发生低甲基化。更重要的是，由这两种致癌物引发的结节中的低甲基化模式相似。此外，在用1,2-DMH或AA引发的结节DNA中，c-myc和c-Ha-ras基因的低甲基化模式总体上也相似。这些结果增加了一种可能性，即肝结节中建立的低甲基化模式可能并不直接反映起始致癌物与DNA之间的相互作用，而可能代表肝结节的一种独特表型。
