Clonal dominance of select subsets of viral kirsten ras+-transformed 3T3 cells during tumor progression

Abstract

Long-term culturing of viral Kirsten-ras-oncogene (v-Ki-ras)-transformed BALB/c 3T3 cells (KiMSV) selectively enriches for cells which have deleted the viral oncogene. In contrast, v-Ki-ras in vivo is amplified and expression increased in all late-stage tumors and lung metastases relative to late-passage KiMSV cells being injected. The nature and significance of these selection processes, in terms of the v-Ki-ras gene, have been explored using genetically-tagged cells, as have the properties of v-Ki-ras- revertant subclones. Inoculation of KiMSV late-passage cells (containing ≤5% v-Ki-ras⁺ cells) into nude mice, generated primary and lung metastatic tumors with the v-Ki-ras gene at increased dosage in all tumors and their single-cell clones, isolated at both early and late stages of tumor development; this demonstrates early and specific in vivo selection for v-Ki-ras⁺ cells in both induction and progression of tumors in this system. v-Ki-ras^- revertant subclones, isolated from late-passage KiMSV cells and inoculated individually into athymic nude mice, yielded tumors for only I of the 4 revertants, with no evidence for v-Ki-ras sequences in these tumor cells, thereby revealing a v-Ki-ras^-independent mechanism for tumor formation in a small subset of revertant cells. Mixtures of the 4 subclones yielded tumors in all animals, although at a much longer latent period than observed with v-Ki-ras⁺ cells. Experiments with mixtures of v-Ki-ras^- revertant cells and pSV2neo-tagged/v-Ki-ras⁺ cells (both complex NeoR cell mixtures and individual Neo clones tested) at various cell ratios revealed clonal variability among v-Ki-ras⁺ cells for dominance during tumor formation. Moreover, the complex Neo cell mixtures yielded both primary and metastatic tumors with simplified patterns of pSV2neo integration sites, suggesting that secondary genetic or epigenetic events, in addition to v-Ki-ras, contribute to the tumor-progressing phenotype. These experiments taken together demonstrate (a) clone-specific early selection of distinct v-Ki-ras⁺ cells amongst themselves and over v-Ki-ras^- cells in both induction and progression of tumors, (b) reduced tumorigenic competence of v-Ki-ras^- revertant cells, with a small subset displaying a v-Ki-ras-independent mechanism for tumor formation in this BALB/c 3T3 system, and (c) the significance of additional genetic or epigenetic events for tumor-progressing competence in unique subsets of v-Ki-ras⁺ cells.