Charles E. Patek

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

ABSTRACT 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.

Mutated K-ras(Asp12) promotes tumourigenesis in Apc(Min) mice more in the large than the small intestines, with synergistic effects between K-ras and Wnt pathways.

K‐ras mutations are found in 40–50% of human colorectal adenomas and carcinomas, but their functional contribution remains incompletely understood. Here, we show that a conditional mutant K‐ras mouse model (K‐rasAsp12/Cre), with transient intestinal Cre activation by β‐Naphthoflavone (β‐NF) treatment, displayed transgene recombination and K‐rasAsp12 expression in the murine intestines, but developed few intestinal adenomas over 2 years. However, when crossed with ApcMin/+ mice, the K‐rasAsp12/Cre/ApcMin/+ offspring showed acceleration of intestinal tumourigenesis with significantly changed average lifespan (P < 0.05) decreased to 18.4 ± 5.4 weeks from 20.9 ± 4.7 weeks (control ApcMin/+ mice). The numbers of adenomas in the small intestine and large intestine were significantly (P < 0.01) increased by 1.5‐fold and 5.7‐fold, respectively, in K‐rasAsp12/Cre/ApcMin/+ mice compared with ApcMin/+ mice, with the more marked increase in adenoma prevalence in the large intestine. To explore possible mechanisms for K‐rasAsp12 and ApcMin co‐operation, the Mitogen‐activated protein kinase (Mapk), Akt and Wnt signalling pathways, including selected target gene expression levels, were evaluated in normal large intestine and large intestinal tumours. K‐rasAsp12 increased activation of Mapk and Akt signalling pathway targets phospho‐extracellular signal‐regulated kinase (pErk) and pAkt, and increased relative expression levels of Wnt pathway targets vascular endothelial growth factor (VEGF), gastrin, cyclo‐oxygenase 2 (Cox2) and T‐cell lymphoma invasion and metastasis 1 (Tiam1) in K‐rasAsp12/Cre/ApcMin/+ adenomas compared with that of ApcMin/+ adenomas, although other Wnt signalling pathway target genes such as Peroxisome proliferator‐activated receptor delta (PPARd), matrix metalloproteinase 7 (MMP7), protein phosphatase 1 alpha (PP1A) and c‐myc remained unchanged. In conclusion, intestinal expression of K‐rasAsp12 promotes mutant Apc‐initiated intestinal adenoma formation in vivo more in the large intestine than the small intestine, with evidence of synergistic co‐operation between mutant K‐ras and Apc involving increased expression of some Wnt‐pathway target genes.

The wt1-heterozygous mouse; a model to study the development of glomerular sclerosis

In the present study, it is shown that mice heterozygous for wt1 develop glomerular sclerosis and the nature and time course of events leading to the glomerular scarring are determined. Wt1‐heterozygous (wt1het) mice and their wild‐type littermates were closely monitored from birth and plasma levels of urea, creatinine, and albumin were compared with histological data and clinical features. One of the first indications of nephropathy in the wt1het mouse was the development of proteinuria, accompanied by progressive elevation of the plasma levels of urea and creatinine. Subsequently, the mice developed albuminuria, which correlated with thickening of the glomerular basement membrane and fusion of the podocyte foot processes. Glomerulosclerosis was a relatively late event, accompanied by severe albuminuria and loss of WT1, nephrin, CD2AP, and α‐actinin‐4. Copyright

Mutant K-ras enhances apoptosis in embryonic stem cells in combination with DNA damage and is associated with increased levels of p19(ARF).

The roles of K-ras in neoplasia are not entirely understood, although there is evidence that K-ras affects susceptibility to apoptosis, modulating survival of DNA damaged cells which would otherwise be eliminated. In this study, we investigated the effects of mutant K-ras on apoptosis in vitro following DNA damage. To avoid complications resulting from mutations in other cancer-related genes and from the presence of endogenous K-ras, we derived K-ras null embryonic stem cells. Expression of either wild-type or mutant K-ras was reconstructed by stable plasmid transfection. The cell lines were treated with etoposide, cisplatin and UV radiation and apoptosis measured flow cytometrically. Mutant K-ras potentiated the effect of etoposide-derived DNA damage by increasing apoptosis, whereas absence of K-ras had the opposite effect. This pattern was similar but less marked with cisplatin, whereas UV yielded no difference in apoptosis with regard to K-ras status, suggesting that the effect of K-ras on apoptosis is dependent on the nature of the DNA damage. To investigate possible mechanisms, we examined the expression of p19ARF mRNA by RT–PCR. Cells expressing mutant K-ras produced elevated levels of p19ARF mRNA, which could link K-ras status with p53 expression and hence susceptibility to DNA damage-induced apoptosis.

Conditional expression of mutated K-ras accelerates intestinal tumorigenesis in Msh2-deficient mice

K-ras mutation occurs in 40–50% of human colorectal adenomas and carcinomas, but its contribution to intestinal tumorigenesis in vivo is unclear. We developed K-rasV12 transgenic mice that were crossed with Ah-Cre mice to generate K-rasV12/Cre mice, which showed β-naphthoflavone-induction of Cre-mediated LoxP recombination that activated intestinal expression of K-rasV12 4A and 4B transcripts and proteins. Only very occasional intestinal adenomas were observed in β-naphthoflavone-treated K-rasV12/Cre mice aged up to 2 years, suggesting that mutated K-ras expression alone does not significantly initiate intestinal tumourigenesis. To investigate the effects of mutated K-ras on DNA mismatch repair (MMR)-deficient intestinal tumour formation, these mice were crossed with Msh2−/− mice to generate K-rasV12/Cre/Msh2−/− offspring. After β-naphthoflavone treatment, K-rasV12/Cre/Msh2−/− mice showed reduced average lifespan of 17.3±5.0 weeks from 26.9±6.8 (control Msh2−/− mice) (P<0.01). They demonstrated increased adenomas in the small intestine from 1.41 (Msh2−/− controls) to 7.75 per mouse (increased fivefold, P<0.01). In the large intestine, very few adenomas were found in Msh2−/− mice (0.13 per mouse) whereas K-rasV12/Cre/Msh2−/− mice produced 2.70 adenomas per mouse (increased 20-fold, P<0.01). Over 80% adenomas from K-rasV12/Cre/Msh2−/− mice showed transgene recombination with expression of K-rasV12 4A and 4B transcripts and proteins. Sequencing of endogenous murine K-ras showed mutations in two out of 10 tumours examined from Msh2−/− mice, but no mutations in 17 tumours from K-rasV12/Cre/Msh2−/− mice. Expression of K-rasV12 in tumours caused activation of the mitogen-activated protein kinase and Akt/protein kinase B signaling pathways, demonstrated by phosphorylation of p44MAPK, Akt and GSK3β, as well as transcriptional upregulation of Pem, Tcl-1 and Trap1a genes (known targets of K-rasV12 expression in stem cells). Thus, mutated K-ras cooperates synergistically with MMR deficiency to accelerate intestinal tumorigenesis, particularly in the large intestine.

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 β-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-β and Wnt signalling pathways in the suppression of pancreatic tumorigenesis in the mouse.

K-ras exon 4A has a tumour suppressor effect on carcinogen-induced murine colonic adenoma formation.

K‐ras encodes two isoforms, K‐ras 4A and 4B, that are jointly affected by K‐ras activating mutations, which are prevalent in colorectal cancer (CRC). CRC shows alterations in the expressed K‐ras 4A : 4B isoform ratio in favour of K‐ras 4B, in tumours both with and without K‐ras mutations. The present study evaluated whether K‐ras 4A expression can suppress colonic adenoma development in the absence of its oncogenic allele. Mice with homozygous targeted deletions of K‐ras exon 4A (K‐rastmΔ4A/tmΔ4A) that can express the K‐ras 4B isoform only, along with heterozygous K‐rastmΔ4A/+ and wild‐type mice, were given ten weekly 1,2‐dimethylhydrazine (DMH) treatments to induce colonic adenomas. There was a significant increase in both the number and the size of colonic adenomas in DMH‐treated K‐rastmΔ4A/tmΔ4A mice, with reduced survival, compared with heterozygous and wild‐type mice. No K‐ras mutations were found in any of the 30 tumours tested from the three groups. Lack of expression of K‐ras 4A transcripts was confirmed, whereas the relative expression levels of K‐ras 4B transcripts were significantly increased in the adenomas of K‐rastmΔ4A/tmΔ4A mice compared with K‐rastmΔ4A/+ and wild‐type mice. Immunohistochemical studies showed that adenomas of K‐rastmΔ4A/tmΔ4A mice had significantly increased cell proliferation and significantly decreased apoptosis with evidence of activation of MapKinase and Akt pathways, with increased phospho‐Erk1/2 and both phospho‐Akt‐Thr308 and phospho‐Akt‐Ser473 immunostaining, compared with adenomas from K‐rastmΔ4A/+ and wild‐type mice. In conclusion, following DMH treatment, K‐ras exon 4A deletion promoted increased number and size of colonic adenomas showing increased K‐ras 4B expression, increased proliferation, decreased apoptosis, and activation of MapKinase and Akt pathways, in the absence of K‐ras mutations. Therefore, K‐ras 4A expression had a tumour suppressor effect on carcinogen‐induced murine colonic adenoma formation, explaining the selective advantage of the altered K‐ras 4A : 4B isoform ratio found in human colorectal cancer. Copyright

Stem cell gene expression changes induced specifically by mutated K-ras.

K-Ras proteins transduce signals from membrane-bound receptors via multiple downstream effector pathways and thereby regulate fundamental stem cell processes that affect neoplasia, including proliferation, apoptosis, and differentiation, but their contribution to tumourigenesis is unclear. Because cancers develop from stem cells, we set out to determine the characteristic changes in gene expression brought about by mutated K-ras (without interference from normal K-ras) in otherwise normal stem cells. cDNA microarrays were used to analyze gene expression profiles comparing wild-type murine embryonic stem (ES) cells with K-ras(Val12) expressing ES cells (previously made null for both endogenous K-ras alleles and transfected with K-ras(Val12), with valine for glycine at codon 12). K-ras(Val12) was expressed at 1.2-fold normal K-ras levels and produced transcripts for both activated K-Ras4A and 4B isoforms. The array expression data were confirmed by real-time quantitative PCR analysis of selected genes expressed both in the K-ras(Val12) expressing ES cells (R = 0.91 with array data) and in the normal intestinal tissues of K-ras(Val12) transgenic mice (R = 0.91 with array data). Changes in gene expression were correlated with the effects of K-ras(Val12) expression on ES cells of enhancing self-renewal in an undifferentiated state, increasing susceptibility to DNA damage-induced apoptosis, and increased proliferation. These expression data may explain, at least in part, some neoplasia-related aspects of the phenotypic changes brought about in this ES cell line by mutated K-ras, in that upregulation of cell growth-related proteins and DNA-associated proteins is consistent with increased proliferation; upregulation of certain apoptosis-related proteins is consistent with a greater susceptibility to DNA damage-induced apoptosis; and downregulation of structural proteins, extracellular matrix components, secretory proteins and receptors is consistent with a less differentiated phenotype.

Age-related changes in the response of chick lens cells during long-term culture to insulin, cyclic AMP, retinoic acid and a bovine retinal extract

We have reported that 1-day-old post-hatch chick lens epithelial cells lose the capacity for lentoid body formation and delta-crystallin expression during long-term serial subculture, although they continue to synthesize, but not to accumulate, alpha- and beta-crystallins, even in cells with a transformed phenotype. Here we present evidence that dedifferentiation may reflect an age-related change in the capacity for response to regulatory signals. We have tested the capacity of these cells in serial subcultures to respond to agencies which affect lens cell growth and differentiation in primary culture: retinoic acid (RA), insulin, cAMP and bovine retinal extract (BRE). Secondary cultures responded only to RA and BRE, by an increase in lentoid formation and by alpha- and beta-accumulation, while RA also restored delta-crystallin expression. Later cultures showed no such responses. The results suggest that the process of lens cell dedifferentiation may, at first, be reversible but later becomes irreversible, despite the continuing persistence of low levels of crystallin expression.

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

BackgroundAlterations 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-rastmΔ4A/tmΔ4A 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-rastmΔ4A/tmΔ4A mice that can, and cannot, express the K-ras 4A proto-oncoprotein respectively.MethodsThe 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-rastmΔ4A/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 Students 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.ResultsThe 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.ConclusionThe 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.