The pro-apoptotic K-Ras 4A proto-oncoprotein does not affect tumorigenesis in the ApcMin/+ mouse small intestine.

BACKGROUND: Alterations in gene splicing occur in human sporadic colorectal cancer (CRC) and may contribute to tumour progression. The K-ras proto-oncogene encodes two splice variants, K-ras 4A and 4B, and K-ras activating mutations which jointly affect both isoforms are prevalent in CRC. Past studies have established that splicing of both the K-ras oncogene and proto-oncogene is altered in CRC in favour of K-ras 4B. The present study addressed whether the K-Ras 4A proto-oncoprotein can suppress tumour development in the absence of its oncogenic allele, utilising the ApcMin/+ (Min) mouse that spontaneously develops intestinal tumours that do not harbour K-ras activating mutations, and the K-rastmDelta4A/tmDelta4A mouse that can express the K-ras 4B splice variant only. By this means tumorigenesis in the small intestine was compared between ApcMin/+, K-ras+/+ and ApcMin/+, K-rastmDelta4A/tmDelta4A mice that can, and cannot, express the K-ras 4A proto-oncoprotein respectively. METHODS: The relative levels of expression of the K-ras splice variants in normal small intestine and small intestinal tumours were quantified by real-time RT-qPCR analysis. Inbred (C57BL/6) ApcMin/+, K-ras+/+ and ApcMin/+, K-rastmDelta4A/tmDelta4A 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 t-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-ras activating mutation. RESULTS: The K-ras 4A transcript accounted for about 50% of K-ras expressed in the small intestine of both wild-type and Min mice. Tumours in the small intestine of Min mice showed increased levels of K-ras 4B transcript expression, but no appreciable change in K-ras 4A transcript levels. No K-ras activating mutations were detected in 27 intestinal tumours derived from Min and compound mutant Min mice. K-Ras 4A deficiency did not affect mouse survival, or tumour number, size or histopathology. CONCLUSION: The K-Ras 4A proto-oncoprotein does not exhibit tumour suppressor activity in the small intestine, even though the K-ras 4A/4B ratio is reduced in adenomas lacking K-ras activating mutations.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Polyploid cells in the mouse ovary

Cell ploidy in the ovarian follicle and corpus luteum was investigated by DNA in situ hybridization to a reiterated, chromosome 3 transgene in mice that were hemizygous for the transgene. This approach was first validated by analysis of mouse kidney, pancreas and liver control tissues, which contain different frequencies of polyploid nuclei. Polyploid nuclei (with multiple hybridization signals) were seen in histological sections of both ovarian follicles and corpora lutea. The frequency of polyploid nuclei in follicles showed no consistent relationship with age (between 6 weeks and 10 months) but polyploid nuclei were significantly more abundant in corpora lutea than follicles (6.3% vs. 2.5%). This implies that production of polyploid cells is more closely associated with differentiation of ovarian follicles into corpora lutea than with the age of the female. Polyploidy tended to be more frequent in corpora lutea of mice that had mated even if they did not become pregnant. This study has highlighted the presence of polyploid cells in the mouse ovarian follicle and corpus luteum and has identified mating as a possible trigger for polyploidy in the corpus luteum. Further work is required to determine the physiological role of polyploid ovarian cells in reproduction

The pro-apoptotic K-Ras 4A proto-oncoprotein does not affect tumorigenesis in the ApcMin/+ mouse small intestine.

BACKGROUND: Alterations in gene splicing occur in human sporadic colorectal cancer (CRC) and may contribute to tumour progression. The K-ras proto-oncogene encodes two splice variants, K-ras 4A and 4B, and K-ras activating mutations which jointly affect both isoforms are prevalent in CRC. Past studies have established that splicing of both the K-ras oncogene and proto-oncogene is altered in CRC in favour of K-ras 4B. The present study addressed whether the K-Ras 4A proto-oncoprotein can suppress tumour development in the absence of its oncogenic allele, utilising the ApcMin/+ (Min) mouse that spontaneously develops intestinal tumours that do not harbour K-ras activating mutations, and the K-rastmDelta4A/tmDelta4A mouse that can express the K-ras 4B splice variant only. By this means tumorigenesis in the small intestine was compared between ApcMin/+, K-ras+/+ and ApcMin/+, K-rastmDelta4A/tmDelta4A mice that can, and cannot, express the K-ras 4A proto-oncoprotein respectively. METHODS: The relative levels of expression of the K-ras splice variants in normal small intestine and small intestinal tumours were quantified by real-time RT-qPCR analysis. Inbred (C57BL/6) ApcMin/+, K-ras+/+ and ApcMin/+, K-rastmDelta4A/tmDelta4A 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 t-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-ras activating mutation. RESULTS: The K-ras 4A transcript accounted for about 50% of K-ras expressed in the small intestine of both wild-type and Min mice. Tumours in the small intestine of Min mice showed increased levels of K-ras 4B transcript expression, but no appreciable change in K-ras 4A transcript levels. No K-ras activating mutations were detected in 27 intestinal tumours derived from Min and compound mutant Min mice. K-Ras 4A deficiency did not affect mouse survival, or tumour number, size or histopathology. CONCLUSION: The K-Ras 4A proto-oncoprotein does not exhibit tumour suppressor activity in the small intestine, even though the K-ras 4A/4B ratio is reduced in adenomas lacking K-ras activating mutations.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Carcinogen-induced pancreatic lesions in the mouse:effect of Smad4 and Apc genotypes

Mutations in the tumour suppressor genes SMAD4 (DPC4, deleted in pancreatic cancer locus 4) and adenomatous polyposis coli (APC) have been implicated in the development of pancreatic cancer in humans. Treatment of wild-type, Smad4+/-, ApcMin/+ or ApcMin/+Smad4+/- mice with N-Nitroso-N-Methyl Urea (NMU) results in abnormal foci in pancreatic acinar cells characterized by increased levels of -catenin. Previously such foci have been shown to be the precursors of pancreatic neoplasia. Interestingly, only NMU-treated ApcMin/+Smad4+/- mice exhibit a significant increase in abnormal pancreas, which was found to be due to increased number of abnormal foci rather than increased focus size. A range of foci sizes were analysed, but only smaller abnormal foci were characterized by morphological nuclear atypia. These studies suggest functional co-operation between TGF- and Wnt signalling pathways in the suppression of pancreatic tumorigenesis in the mouse

Repeated Evolution of Power-Amplified Predatory Strikes in Trap-Jaw Spiders

Small animals possess intriguing morphological and behavioral traits that allow them to capture prey, including innovative structural mechanisms that produce ballistic movements by amplifying power [1-6]. Power amplification occurs when an organism produces a relatively high power output by releasing slowly stored energy almost instantaneously, resulting in movements that surpass the maximal power output of muscles [7]. For example, trap-jaw, power-amplified mechanisms have been described for several ant genera [5, 8], which have evolved some of the fastest known movements in the animal kingdom [6]. However, power-amplified predatory strikes were not previously known in one of the largest animal classes, the arachnids. Mecysmaucheniidae spiders, which occur only in New Zealand and southern South America, are tiny, cryptic, ground-dwelling spiders that rely on hunting rather than web-building to capture prey [9]. Analysis of high-speed video revealed that power-amplified mechanisms occur in some mecysmaucheniid species, with the fastest species being two orders of magnitude faster than the slowest species. Molecular phylogenetic analysis revealed that power-amplified cheliceral strikes have evolved four times independently within the family. Furthermore, we identified morphological innovations that directly relate to cheliceral function: a highly modified carapace in which the cheliceral muscles are oriented horizontally; modification of a cheliceral sclerite to have muscle attachments; and, in the power-amplified species, a thicker clypeus and clypeal apodemes. These structural innovations may have set the stage for the parallel evolution of ballistic predatory strikes

Murine Denys-Drash syndrome: evidence of podocyte de-differentiation and systemic mediation of glomerulosclerosis

Denys-Drash syndrome (DDS) is caused by dominant mutations of the Wilms' tumour suppressor gene, WT1, and characterized by a nephropathy involving diffuse mesangial sclerosis, male pseudohermaphroditism and/ or Wilms' tumourigenesis. Previously, we reported that heterozygosity for the Wt1tmT396 mutation induces DDS in heterozygous and chimeric (Wt1tmT396/+ ↔ +/+) mice. In the present study, the fate of Wt1 mutant cells in chimeric kidneys was assessed by in situ marker analysis, and immunocytochemistry was used to re-examine the claim that glomerulosclerosis (GS) is caused by loss of WT1 and persistent Pax-2 expression by podocytes. Wt1 mutant cells colonized glomeruli efficiently, including podocytes, but some sclerotic glomeruli contained no detectable Wt1 mutant cells. The development of GS was preceded by widespread loss of ZO-1 signal in podocytes (even in kidneys wher

While K-ras Is Essential for Mouse Development, Expression of the K-ras 4A Splice Variant Is Dispensable

In mammals, the three classical ras genes encode four highly homologous proteins, N-Ras, H-Ras, and the isoforms K-Ras 4A and 4B. Previous studies have shown that K-ras is essential for mouse development and that while K-ras 4A and 4B are expressed during development, K-ras 4A expression is regulated temporally and spatially and occurs in adult kidney, intestine, stomach, and liver. In the present study, the pattern of K-ras 4A expression was examined in a wide range of wild-type adult mouse tissues, and gene targeting was used to generate K-ras 4A-deficient mice to examine its role in development. It was found that K-ras 4A is also expressed in uterus, lung, pancreas, salivary glands, seminal vesicles, bone marrow cells, and cecum, where it was the major K-Ras isoform expressed. Mating between K-ras(tmΔ4A/+) mice produced viable K-ras(tmΔ4A/tmΔ4A) offspring with the expected Mendelian ratios of inheritance, and these mice expressed the K-ras 4B splice variant only. K-ras(tmΔ4A/tmΔ4A) mice were fertile and showed no histopathological abnormalities on inbred (129/Ola) or crossbred (129/Ola × C57BL/6) genetic backgrounds. The results demonstrate that K-Ras 4A, like H- and N-Ras, is dispensable for normal mouse development, at least in the presence of functional K-Ras 4B