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Clonal Diversity of the Kirsten-ras Oncogene during Tumor Progression in Athymic Nude Mice: Mechanisms of Amplification and Rearrangement¹

Department of Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106 [R. R., L. A. C.]; Los Alamos National Laboratory, Experimental Pathology Group LS-4, Los Alamos, New Mexico 87545 [P. M. K.]; and Department of Immunology and Cancer Research, Cleveland Clinic Foundation, Cleveland, Ohio 44106 [M. R. P.]

Single-cell clones from primary and lung metastatic tumors have been evaluated for the state of the viral-Kirsten-ras oncogene (v-Ki-ras) by Southern blot analysis after injection of Kirsten sarcoma virus-transformed BALB/c 3T3 cells (KiMSV, with a replication-defective provirus) into athymic nude mice by four different injection routes. While all clones of early-passage KiMSV cells contained an EcoRI-generated 5.3-kilobase DNA fragment at high dosage level, most clones of late-passage cells had lost this v-Ki-ras fragment or had greatly diminished levels. However, all clones of all tumors (>90 tested) obtained after injection of these latepassage cells contained a dosage of the 5.3-kilobase v-Ki-ras band similar to that of the early-passage KiMSV cells, suggesting either a very strong selection for v-Ki-ras-bearing cells of the early-passage type in tumor formation and/or the ability of a subset of late-passage cells to amplify this gene to some minimal dosage level. Both flow cytometric analyses for DNA content and quantitation of chromosomes showed that all primary and lung metastatic tumors had more than twice the number of chromosomes as the late-passage KiMSV cells; however, four of 80 latepassage cells had a chromosome count in the range of tumors, consistent with their importance in tumor generation and possibly amplification of the v-Ki-ras-bearing chromosome. Clonal analyses of lung micrometastatic tumors revealed a v-Ki-ras blot pattern identical to that of the s.c. primary tumors. However, two of five lung metastases from the footpad (as large rapidly growing nodules) and i.v. routes had multiple copies of v-Ki-ras in new sites; a second injection round led to even greater complexity in v-Ki-ras patterns in clones of lung tumors. Two assays were used to demonstrate that these new v-Ki-ras integrations were generated by superinfection with a "helper" retrovirus, not sarcomagenic by itself in the nude mice, that led to rescue/reinfection of tumor cells with the defective Kirsten sarcoma proviral genome—cellular transformation of 3T3 or C3H10T1/2 cells and RNA dot blot analyses for medium-secreted retrovirus specific for LTR or v-Ki-ras sequences. This "helper" retrovirus could not be detected in early-or late-passage KiMSV cells used for inoculation but could be detected in certain tissues of normal nude mice, demonstrating its in vivo origin. In summary, clonal analyses of the v-Ki-ras oncogene in both primary and lung metastatic tumor populations have suggested mechanisms for amplification and rearrangement of this oncogene during tumor progression in this system and provide the first evidence for synergy between a nude mouse-borne retrovirus in generating new insertion sites for this oncogene, making them more highly metastatic in the lung site.

¹ Supported by NIH Research Grant CA27755 (L. A. C.) and the Department of Energy (P. M. K.). Case Western Reserve University is a comprehensive cancer center supported by USPHS Grant P30CA43703, awarded by the National Cancer Institute, Department of Health and Human Services.

² To whom requests for reprints should be addressed.

Received 3/17/88. Revised 5/25/88. Accepted 6/ 3/88.

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