The pro-apoptotic K-Ras 4A proto-oncoprotein does not affect tumorigenesis in the <it>Apc</it>^Min/+mouse small intestine

• Main Authors: Berry Rachel L, Devenney Paul S, Walker Marion, Luo Feijun, Rose Lorraine, Arends Mark J, Patek Charles E, Lawrence Nicola J, Ridgway Rachel A, Sansom Owen J, Hooper Martin L
• Format: Article
• Language: English
• Published: BMC 2008-06-01
• Series: BMC Gastroenterology
• Online Access: http://www.biomedcentral.com/1471-230X/8/24
• ISSN: 1471-230X

<p>Abstract</p> <p>Background</p> <p>Alterations in gene splicing occur in human sporadic colorectal cancer (CRC) and may contribute to tumour progression. The K-<it>ras </it>proto-oncogene encodes two splice variants, K-<it>ras </it>4A and 4B, and K-<it>ras </it>activating mutations which jointly affect both isoforms are prevalent in CRC. Past studies have established that splicing of both the K-<it>ras </it>oncogene and proto-oncogene is altered in CRC in favour of K-<it>ras </it>4B. The present study addressed whether the K-Ras 4A proto-oncoprotein can suppress tumour development in the <it>absence </it>of its oncogenic allele, utilising the <it>Apc</it>^Min/+(<it>Min</it>) mouse that spontaneously develops intestinal tumours that do not harbour K-<it>ras </it>activating mutations, and the K-<it>ras</it>^{tmΔ4A/tmΔ4A}mouse that can express the K-<it>ras </it>4B splice variant only. By this means tumorigenesis in the small intestine was compared between <it>Apc</it>^Min/+, K-<it>ras</it>^+/+and <it>Apc</it>^Min/+, K-<it>ras</it>^{tmΔ4A/tmΔ4A}mice that can, and cannot, express the K-<it>ras </it>4A proto-oncoprotein respectively.</p> <p>Methods</p> <p>The relative levels of expression of the K-<it>ras </it>splice variants in normal small intestine and small intestinal tumours were quantified by real-time RT-qPCR analysis. Inbred (C57BL/6) <it>Apc</it>^Min/+, K-<it>ras</it>^+/+and <it>Apc</it>^Min/+, K-<it>ras</it>^{tmΔ4A/tmΔ4A}mice were generated and the genotypes confirmed by PCR analysis. Survival of stocks was compared by the Mantel-Haenszel test, and tumour number and area compared by Student's <it>t</it>-test in outwardly healthy mice at approximately 106 and 152 days of age. DNA sequencing of codons 12, 13 and 61 was performed to confirm the intestinal tumours did not harbour a K-<it>ras </it>activating mutation.</p> <p>Results</p> <p>The K-<it>ras </it>4A transcript accounted for about 50% of K-<it>ras </it>expressed in the small intestine of both wild-type and <it>Min </it>mice. Tumours in the small intestine of <it>Min </it>mice showed increased levels of K-<it>ras </it>4B transcript expression, but no appreciable change in K-<it>ras </it>4A transcript levels. No K-<it>ras </it>activating mutations were detected in 27 intestinal tumours derived from <it>Min </it>and compound mutant <it>Min </it>mice. K-Ras 4A deficiency did not affect mouse survival, or tumour number, size or histopathology.</p> <p>Conclusion</p> <p>The K-Ras 4A proto-oncoprotein does not exhibit tumour suppressor activity in the small intestine, even though the K-<it>ras </it>4A/4B ratio is reduced in adenomas lacking K-<it>ras </it>activating mutations.</p>