Lu D, Kunz H W, Melhem M F, Gill T J
Department of Pathology, University of Pittsburgh, School of Medicine, Pennsylvania 15261.
Cancer Res. 1993 Sep 1;53(17):4089-95.
The growth and reproduction complex (grc-) strains of rats have a 70-kilobase deletion in the major histocompatibility complex (MHC)-linked grc-G/C region that is associated with embryonic death, developmental defects, and an increased susceptibility to chemical carcinogens. To study further the effects associated with the deletion, fibroblastic cell lines from grc-, grc+, and grc+/- rat embryos were developed: BIL-derived cell lines are congenic for the MHC and grc, whereas R16-derived cell lines are congenic for the grc alone. In early passages, all cell lines expressed the MHC class I antigen RT1.A, had a diploid chromosome number, and did not display anchorage-independent growth or in vivo tumorigenicity. The grc- cells [median population doubling time (PDT), 47 h] grew more slowly than the grc+ (PDT, 30.5 h) and grc+/- (PDT, 33 h) cells. All cells underwent crisis, but the crisis stage began earlier and lasted longer in the grc- cells. The established grc- cell lines (PDT, 32.5 h) grew faster than the grc+ (PDT, 48.5 h) and grc+/- (PDT, 54 h) cell lines. Two of the three BIL-derived grc- lines that survived crisis became anchorage independent in tissue culture and tumorigenic in histocompatible F1 rats (highly malignant fibrosarcomas) at passages 33 and 48, respectively; by contrast, none of the R16-derived grc- cell lines transformed. None of 8 grc+ or 8 grc+/- cell lines that survived crisis displayed anchorage-independent growth or tumorigenicity under the same conditions up to passage 50. All of the established cell lines, including the two tumorigenic ones, expressed MHC class I antigens. Southern and Northern blot analyses of BIL-derived cell lines before and after crisis showed that they all constitutively expressed H-ras and Rb and that no cell line showed rearrangement, amplification, or overexpression of c-myc, H-ras, Rb, and p53 either before or after crisis. These observations indicate that: (a) the homozygous grc- deletion is necessary but not sufficient for in vitro transformation; (b) another genetic factor(s) required for transformation is linked to, or possibly in, the MHC; and (c) passage through crisis, spontaneous transformation, or carcinogen treatment does not alter the cellular expression of MHC class I antigens or of several oncogenes and tumor suppressor genes.
大鼠的生长与繁殖复合体（grc-）品系在主要组织相容性复合体（MHC）连锁的grc-G/C区域有一个70千碱基的缺失，这与胚胎死亡、发育缺陷以及对化学致癌物易感性增加有关。为了进一步研究与该缺失相关的影响，构建了来自grc-、grc+和grc+/-大鼠胚胎的成纤维细胞系：源自BIL的细胞系在MHC和grc方面是同源的，而源自R16的细胞系仅在grc方面是同源的。在早期传代时，所有细胞系均表达MHC I类抗原RT1.A，具有二倍体染色体数，并且不表现出不依赖贴壁生长或体内致瘤性。grc-细胞[群体倍增时间（PDT）中位数为47小时]比grc+（PDT为30.5小时）和grc+/-（PDT为33小时）细胞生长得更慢。所有细胞都经历了危机期，但危机期在grc-细胞中开始得更早且持续时间更长。已建立的grc-细胞系（PDT为32.5小时）比grc+（PDT为48.5小时）和grc+/-（PDT为54小时）细胞系生长得更快。在危机期存活下来的源自BIL的三个grc-细胞系中有两个在组织培养中变得不依赖贴壁生长，并分别在第33代和第48代时在组织相容性F1大鼠中具有致瘤性（高恶性纤维肉瘤）；相比之下，源自R16的grc-细胞系均未发生转化。在危机期存活下来的8个grc+或8个grc+/-细胞系在传代至50代之前，在相同条件下均未表现出不依赖贴壁生长或致瘤性。所有已建立的细胞系，包括两个具有致瘤性的细胞系，均表达MHC I类抗原。对源自BIL的细胞系在危机期前后进行的Southern和Northern印迹分析表明，它们均组成性表达H-ras和Rb，并且在危机期前后没有任何细胞系显示c-myc、H-ras、Rb和p53发生重排、扩增或过表达。这些观察结果表明：（a）纯合的grc-缺失对于体外转化是必要的，但不是充分的；（b）转化所需的另一个遗传因素与MHC连锁，或可能存在于MHC中；（c）经历危机期、自发转化或致癌物处理不会改变MHC I类抗原以及几种癌基因和肿瘤抑制基因的细胞表达。
