Amplification and rearrangement of the Kirsten ras oncogene in virus-transformed BALB/c 3T3 cells during malignant tumor progression.

Abstract

Analyses of the cellular and viral Kirsten ras genes (c-Ki-ras and v-Ki-ras, respectively) Ki-ras led to no further rearrangements in the lung microfoci tumors isolated >40 days after injection. These data suggest (i) the significance of amplification and elevated expression of v-Ki-ras in tumor formation, (ii) correlation of this ampliflication with more effective tumor progression, and (iii) the selective advantage that cells with Ki-ras DNA sequence additions have in the formation of overt lung tumors.During tumor progression many changes may occur genotypically (1) and phenotypically (2), including increases in gene dosage and expression of oncogenes (3). The ras oncogene has special importance in select tumors. Harvey and Kirsten ras oncogenes were discovered as the transforming component of murine sarcoma retroviruses (4, 5), and N-ras was found in human DNA (3). DNA transfection experiments and isolation ofras genes from numerous tumors established that activation ofthe cellular ras protooncogene's transforming and tumorigenic potential occurs either by a point mutation replacing amino acids at position 12 or 61 in the encoded 21-kDa membrane protein (6) or by increasing the amount of the ras gene and hence its product (7). ras oncogenes can enhance transformation and tumorigenicity in established cell lines, but in primary cells a second oncogene is required with some exceptions (8).Although ras has been analyzed in primary tumor cells, much less is known about its state in metastatic populations. ras is of particular interest because of recent evidence pointing to its involvement in malignant tumor progression.Transfection with tumor DNA (9, 10) or activated ras oncogenes (11-13) demonstrated that the metastatic phenotype can be transferred between cells (14). ras DNA and RNA levels in NIH 3T3 transfectants and N-nitroso-Nmethylurea-induced tumors containing an activated cellular Harvey ras protooncogene (c-Ha-ras) showed gene amplification and increased expression in primary tumors with comparable levels in the metastatic lesions (14). These data suggest a role for ras in malignant tumor progression.These data prompted analyses of the Kirsten ras oncogene during tumor progression using a Kirsten murine sarcoma virus (KiMSV)-transformed BALB/p 3T3 cell line that harbors a replication-defective viral Kirsten ras oncogene (vKi-ras)-containing provirus (15). Cells were injected into athymic nude mice in four anatomical locations. The resulting primary tumors and lung metastases were isolated, adapted to in vitro growth, and analyzed for DNA levels and expression of v-Ki-ras and the cellular Kirsten ras protooncogene (c-Ki-ras) relative to the untransformed BALB/c 3T3 and KiMSV (parental) transformed cells. Analyses of lung metastases undergoing a second round of in vivo selection using specific tumor populations from the first round of injections were also performed. MATERIALS AND METHODSCells and Tumors. BALB/c 3T3 cells (clone A31) and KiMSV-transformed BALB/c 3T3 cells (nonproducer clone k-234...

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