Altered Growth of Human Colon Cancer Cell Lines Disrupted at Activated Ki- ras

Abstract

Point mutations that activate the Ki-ras proto-oncogene are presented in about 50 percent of human colorectal tumors. To study the functional significance of these mutations, the activated Ki-ras genes in two human colon carcinoma cell lines, DLD-1 and HCT 116, were disrupted by homologous recombination. Compared with parental cells, cells disrupted at the activated Ki-ras gene were morphologically altered, lost the capacity for anchorage-independent growth, grew more slowly both in vitro and in nude mice, and showed reduced expression of c-myc. Thus, the activated Ki-ras gene plays a key role in colorectal tumorigenesis through altered cell differentiation and cell growth.

Low-dose metronomic cyclophosphamide treatment mediates ischemia-dependent K-ras mutation in colorectal carcinoma xenografts

“…While originally reported to be non-tumorigenic (Shirasawa et al, 1993) in our hands these cells demonstrate attenuated tumorigenicity (Shahrzad et al, 2005), and in this study formed palpable tumors in all mice 45 days after subcutaneous injection of 2 Â 10 6 cells into the right flank of RAG-1 null mice. When the average tumor size reached 35 mm 3 mice were randomized into treatment and control groups and subjected to low-dose (metronomic) cyclophosphamide (LDM) for a period of 2 weeks followed by a 2-week break period.…”

“…Accelerated tumor growth post-metronomic (LDM) therapy Dks-8 cells are K-ras wt/null (Shirasawa et al, 1993), microsatellite unstable and deficient for the MMR enzyme MSH6 (Boyer et al, 1995). While originally reported to be non-tumorigenic (Shirasawa et al, 1993) in our hands these cells demonstrate attenuated tumorigenicity (Shahrzad et al, 2005), and in this study formed palpable tumors in all mice 45 days after subcutaneous injection of 2 Â 10 6 cells into the right flank of RAG-1 null mice.…”

“…The system employed in this study uses a MMRdeficient CRC cell line that has been artificially engineered to disrupt a previously mutant K-ras allele (Shirasawa et al, 1993). Since K-ras mutation is known to lead to other alterations in cancer cell behavior contributing to tumor progression (notably uncontrolled proliferation and enhanced production of angiogenic factors; Bos, 1989;Rak et al, 1995), it is possible that CRC cells that were always wild type for K-ras may behave differently under similar experimental conditions.…”

“…We found no evidence for de novo development of mutation at this additional codon in either control or treatment group of the xenografted tumors (Supplementary Figure 1). To originally generate the K-ras wt/null cell line Dks-8, DLD-1 mutant K-ras was selectively disrupted by homologous recombination, a method that interferes with the detection of de novo development of mutation at codon 12 (Shirasawa et al, 1993); thus we could not test the xenografts for changes at this codon.…”

“…DLD-1 K-ras G13D was selectively disrupted by homologous recombination to generate the K-ras wt/null cell line Dks-8 (Shirasawa et al, 1993). Both DLD-1 and Dks-8 cells are microsatellite-unstable and deficient for the MMR enzyme MSH6 (Boyer et al, 1995).…”