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-ras(V12) transgenic mice that were crossed with Ah-Cre mice to generate K-ras(V12)/Cre mice, which showed beta-naphthoflavone-induction of Cre-mediated LoxP recombination that activated intestinal expression of K-ras(V12) 4A and 4B transcripts and proteins. Only very occasional intestinal adenomas were observed in beta-naphthoflavone-treated K-ras(V12)/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-ras(V12)/Cre/Msh2(-/-) offspring. After beta-naphthoflavone treatment, K-ras(V12)/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-ras(V12)/Cre/Msh2(-/-) mice produced 2.70 adenomas per mouse (increased 20-fold, P<0.01). Over 80% adenomas from K-ras(V12)/Cre/Msh2(-/-) mice showed transgene recombination with expression of K-ras(V12) 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-ras(V12)/Cre/Msh2(-/-) mice. Expression of K-ras(V12) in tumours caused activation of the mitogen-activated protein kinase and Akt/protein kinase B signaling pathways, demonstrated by phosphorylation of p44MAPK, Akt and GSK3beta, as well as transcriptional upregulation of Pem, Tcl-1 and Trap1a genes (known targets of K-ras(V12) expression in stem cells). Thus, mutated K-ras cooperates synergistically with MMR deficiency to accelerate intestinal tumorigenesis, particularly in the large intestine.

K-ras 4A and 4B are co-expressed widely in human tissues, and their ratio is altered in sporadic colorectal cancer.

Ras activating mutations result in constitutive activation of Ras signalling pathways and occur in 30% of human malignancies. K-ras encodes two splice variants, K-ras 4A and 4B, and K-ras activating mutations which jointly affect both isoforms are prevalent in lung, pancreatic and colorectal cancers. Using RT-PCR we examined their expression in normal adult human tissues and addressed whether K-ras splicing is altered in sporadic colorectal cancer by comparing normal colon with colon carcinoma cell lines, and 'matched' tumour and tumour-free colon tissues from the same patient. K-ras 4B was expressed ubiquitously and was the predominant splice variant. K-ras 4A was expressed differentially, with detection in colorectal tumours and cell lines, and normal colon, pancreas and lung--sites where tumours with K-ras activating mutations arise. Both K-ras splice variants were co-expressed by single colon carcinoma cells. The K-ras 4A/4B ratio was significantly reduced in all 6 cell lines examined, including two that lacked K-ras activating mutations, and in 4/9 primary adenocarcinomas. We conclude that K-ras activating mutations do not affect K-ras splicing per se, both isoforms may play a role in neoplastic progression, and altered splicing of either the K-ras proto-oncogene or oncogene, in favour of K-ras 4B, may modulate tumour development.

Gonadal effects of a mouse Denys-Drash syndrome mutation.

Gonadal effects of the Denys-Drash syndrome (DDS) mutation Wt1(tmT396 )were examined in chimaeric and heterozygous mice. Since the only heterozygote was 41,XXY, Sertoli cell function was assessed by comparison with age-matched control XXY testes. Control XXY Sertoli cells showed immuno-expression of WT1 and androgen receptor (AR) indistinguishable from wild-type (40,XY), but expressed anti-Mullerian hormone (AMH). In contrast, DDS Sertoli cells showed only faint immuno-expression of WT1 and did not express AR or AMH. While XY<-->XY DDS chimaeras were male, XX<-->XY chimaeras were predominantly female. In the rare XX<-->XY DDS males the Sertoli cell lineage was largely derived from Wt1 mutant XY cells. We conclude that DDS mutant cells can form Sertoli cells, that the dominant mutation does not cause male sex reversal in mice but distorts the sex ratio of XX<-->XY chimaeras, and that there may be a link between WT1, AMH and AR expression by Sertoli cells in vivo.

C-terminal truncation of WT1 delays but does not abolish hematopoiesis in embryoid bodies.

The effect of mutations that truncate the WT1 protein on in vitro hematopoietic differentiation from embryonal stem cells has been examined by CFU-A assay, o-dianisidine staining for heme, and RT-PCR analysis of the expression of fetal and adult globins. In two independently isolated ES cell lines the mutations delay but do not abolish hematopoiesis. Analysis of replated CFU-A colonies indicates that the delay occurs prior to the formation of hematopoietic stem cells. The results demonstrate a role for WT1 at the onset of hematopoiesis.

Truncation of WT1 results in downregulation of cyclin G1 and IGFBP-4 expression.

Mutations in the WT1 gene are found in a subset of Wilms' tumours and in certain other disorders such as Denys-Drash syndrome. The WT1 gene product is a zinc finger transcription factor for which many target genes have been suggested. Here we utilise gene targeting to generate cells containing only truncated forms of WT1, in which the DNA-binding region is disrupted. Examination of gene expression in these cells using cDNA macroarrays suggests two novel WT1 transcriptional targets, cyclin G1 (Ccng1), and insulin-like growth factor binding protein 4 (Igfbp4).

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 p19(ARF) mRNA by RT-PCR. Cells expressing mutant K-ras produced elevated levels of p19(ARF) mRNA, which could link K-ras status with p53 expression and hence susceptibility to DNA damage-induced apoptosis.

A zinc finger truncation of murine WT1 results in the characteristic urogenital abnormalities of Denys-Drash syndrome.

The Wilms tumor-suppressor gene, WT1, plays a key role in urogenital development, and WT1 dysfunction is implicated in both neoplastic (Wilms tumor, mesothelioma, leukemias, and breast cancer) and nonneoplastic (glomerulosclerosis) disease. The analysis of diseases linked specifically with WT1 mutations, such as Denys-Drash syndrome (DDS), can provide valuable insight concerning the role of WT1 in development and disease. DDS is a rare childhood disease characterized by a nephropathy involving mesangial sclerosis, XY pseudohermaphroditism, and/or Wilms tumor (WT). DDS patients are constitutionally heterozygous for exonic point mutations in WT1, which include mutations predicted to truncate the protein within the C-terminal zinc finger (ZF) region. We report that heterozygosity for a targeted murine Wt1 allele, Wt1(tmT396), which truncates ZF3 at codon 396, induces mesangial sclerosis characteristic of DDS in adult heterozygous and chimeric mice. Male genital defects also were evident and there was a single case of Wilms tumor in which the transcript of the nontargeted allele showed an exon 9 skipping event, implying a causal link between Wt1 dysfunction and Wilms tumorigenesis in mice. However, the mutant WT1(tmT396) protein accounted for only 5% of WT1 in both heterozygous embryonic stem cells and the WT. This has implications regarding the mechanism by which the mutant allele exerts its effect.

Changes in crystallin expression during transdifferentiation and subsequent ageing of embryonic chick neural retina in vitro: comparison with lens epithelium.

We have shown that cultured day-old chick lens epithelial cells undergo changes in crystallin expression during lens fibre (lentoid body) differentiation and ageing in serial subculture which are similar to those found in the adult lens in vivo. Here we have maintained neural retina cells, which transdifferentiate in vitro, for 250 days in serial subculture, in order to determine whether the tissue of origin affects the sequence of changes in crystallin expression and capacity for lentoid body formation both during fibre formation in primary culture and ageing in vitro. Alpha and beta-crystallins were detectable before delta-crystallin in primary cultures of both day-old chick lens epithelium and neural retina from 8-day embryos, but while beta 2 (26 kDa) was detected in pre-lentoid lens epithelial cultures it was not detected until after lentoids formed in neural retina cultures. The relative proportions of the alpha- and beta-crystallin polypeptides were similar in lentoid-rich lens epithelium and neural retina cultures, and both cultures underwent similar changes in serial subculture: a loss of lentoid forming capacity, an early preferential loss of delta-crystallin expression followed by a failure to accumulate first alpha- and then beta-crystallins. The order in which the beta-crystallin polypeptides were lost differed between the cultures. There is evidence for rapid turnover of alpha- and beta-crystallins while actin is the major component expressed in both types of aged cultures. Thus lens and retinal cells show some initial differences in the sequence of crystallin expression in primary cultures and in the eventual characteristics of aged cultures, but during the period beginning with lentoid formation and ending with the onset of senescence, lens cells from either source follow a broadly similar programme of ageing changes which are similar to those which occur during lens ageing in vivo.

Sex chimaerism, fertility and sex determination in the mouse.

Adult intraspecific mouse chimaeras, derived by introducing male embryonal stem cells into unsexed host blastocysts, were examined to determine whether gonadal sex was correlated with the sex chromosome composition of particular cell lineages. The fertility of XX in equilibrium XY and XY in equilibrium XY male chimaeras was also compared. The distribution of XX and XY cells in 34 XX in equilibrium XY ovaries, testes and ovotestes was determined by in situ hybridisation using a Y-chromosome-specific probe. Both XX and XY cells were found in all gonadal somatic tissues but Sertoli cells were predominantly XY and granulosa cells predominantly XX. The sex chromosome composition of the tunica albuginea and testicular surface epithelium could not, in general, be fully resolved, owing to diminished hybridisation efficiency in these tissues, but the ovarian surface epithelium (which like the testicular surface epithelium derives from the coelomic epithelium) was predominantly XX. These findings show that the claim that Sertoli cells were exclusively XY, on which some previous models of gonadal sex determination were based, was incorrect, and indicate instead that in the mechanism of Sertoli cell determination there is a step in which XX cells can be recruited. However, it remains to be established whether the sex chromosome constitution of the coelomic epithelium lineage plays a causal role in gonadal sex determination. Male chimaeras with XX in equilibrium XY testes were either sterile or less fertile than chimaeras with testes composed entirely of XY cells. This impaired fertility was associated with the loss of XY germ cells in atrophic seminiferous tubules. Since this progressive lesion was correlated with a high proportion of XX Leydig cells, we suggest that XX Leydig cells are functionally defective, and unable to support spermatogenesis.

Hypoxanthine phosphoribosyl transferase deficiency, haematopoiesis and fertility in the mouse.

We have looked for effects of deficiency in hypoxanthine phosphoribosyl transferase (HPRT) in the mouse comparable to non-behavioural consequences of HPRT-deficiency in humans. HPRT-deficient humans show abnormalities in haematopoiesis and, in heterozygotes, there is strong selection in haematopoietic tissues against HPRT-deficient cells arising as a result of X-chromosome inactivation. We have examined two situations in mice in which HPRT- and HPRT+ cells occur in the same individual. First, in chimaeras resulting from the injection of HPRT- embryonal stem cells into HPRT+ blastocysts the fate of HPRT- and HPRT+ cell populations was monitored by their expression of different isozymes of glucose phosphate isomerase and also, in those chimaeras that resulted from injecting the male ES cells into female blastocysts, by in situ hybridisation using a Y-chromosome-specific repetitive DNA probe. There was a small statistically significant selection against the HPRT- population in haematopoietic tissues in both XX in equilibrium with XY and XY in equilibrium with XY chimaeras. Second, in female mice doubly heterozygous for HPRT-deficiency and for an electrophoretic variant of the X-linked enzyme phosphoglycerate kinase, there was a similar small statistically significant selection against the HPRT- population in haematopoietic tissues. While further work is required to establish whether this selection is a consequence of the HPRT mutation, it is clear that any selection against cells in the haematopoietic system as a consequence of HPRT-deficiency is at most small compared with the effect seen in humans. In HPRT-deficient human males surviving beyond the normal age of puberty, there is testicular atrophy. However, we find no effect of HPRT-deficiency on the fertility of either male or female mice. Thus, as with effects on behaviour, the consequences of HPRT-deficiency for haematopoiesis and testis development in the mouse are at most small compared with those in the human. We conclude that the reason for the difference in effects between the two species lies in a difference in purine-related intermediary metabolism per se, rather than in its interaction with brain amine biochemistry.

Ageing in the chick lens: in vitro studies.

In principle, ageing may be due to the interaction of several factors, including the accumulation of random changes both genomic and non-genomic, secondary changes in a tissue contingent upon the changing function of other tissues, and programmed non-random changes in the tissue-specific expression of various genes. The use of a single tissue comprising one cell type only, in which the major gene products are well defined, in which there is a well attested series of developmental and age-related changes in cell properties and gene expression and which can be studied and compared in vivo and in vitro, offers advantages for investigation of these questions. The vertebrate eye lens possesses these advantages. The crystallins (proteins expressed at super-abundant levels in the lens) are well characterised. The lens epithelial cells (LEC) grow readily and can differentiate into the lens fibre cells in vitro, and, finally, such terminally differentiated cells may also be derived, by a process of transdifferentiation, from neural retina cells (NRC) in vitro. Thus the effect on ageing changes of the tissue of origin may also be studied. This article reviews our previous studies on long-term changes in growth potential, differentiation capacity and crystallin expression of chick lens cells in ageing cultures, their overall similarity to events in vivo and the effect on ageing changes of genotypes affecting the growth rate. It presents new information on these genetic aspects, and on crystallin expression in long-term ageing cultures of transdifferentiated neural retina, and compares the behaviour of ageing chick lens cells with that reported for mammals.

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.

A close relationship between microvilli and metabolic cooperation deficiency in embryonal carcinoma cells.

A revertant clone has been isolated from the metabolic cooperation-deficient embryonal carcinoma clone PT2md1. In PT2md1, its cooperation-competent parent, and its cooperation-competent revertant an inverse correlation has been demonstrated between cooperation competence and the incidence of microvilli. This demonstration, together with a similar correlation previously reported in an independently isolated series of cell lines, establishes a close causal relationship between the incidence of microvilli and communication deficiency.

The influence of the genotype on the process of ageing of chick lens cells in vitro.

We reported previously that changes in crystallin expression in differentiating long-term primary cultures of lens cells from five different chick genotypes are similar to those which occur in vivo between hatching and the 8-week-old adult. These changes followed a similar program in all genotypes but occurred more rapidly in cells from the fast-growing than from the slow-growing genotypes. The present study examines ageing changes in lens cell populations from the same five genotypes, over a 4-6 month period, using long-term serial subcultures. The capacity for lentoid differentiation was progressively lost, but the rate of loss was inversely related to the intrinsic growth rate of the cells of these genotypes, occurring at the first passage in the slowest-growing strain, while fifth passage cells of the fastest-growing strain still retained some lentoid-forming capacity. The rate of loss of crystallin expression was also inversely related to the genetic growth rate, but the sequence of changes appears to be nonrandom, since it was broadly similar in all genotypes, starting with a preferential loss of delta-crystallin, as occurs in vivo; although alpha- and beta-crystallins were undetectable in late dedifferentiated cultures, the capacity of the cells for their synthesis was still present. Cultures from both fast-growing genotypes eventually showed senescence, but those from all three slow-growing genotypes underwent transformation. The major cell component in late cultures of all genotypes was actin.

Patterns of crystallin expression during differentiation in vitro of several chick genotypes with different effects on lens cell growth rate.

We have recently reported that chick lens cells during differentiation in long-term culture show a programme of change in crystallin expression which mimics events during lens development in vivo. The aim of the present work was to examine the stability of the programme by testing the response to genetic influences and exposure to a carcinogen. Five genetically distinct inbred strains of chick, differing in the intrinsic growth rates of lens epithelial cells in vitro, were used to study the effects of the rate of mitosis on crystallin expression, both during lens development and in long-term cell culture. The time of appearance of lentoids, their size and abundance and the rate of change in crystallin expression were all modified in a genotype-specific way, related to the rate of mitosis, but the programme of changes in crystallin expression was the same for all genotypes. Genetic differences were also found in the patterns of response to treatment of cultures during the logarithmic growth phase with a nitrosoguanidine compound known to affect cell differentiation in lens cultures and several other systems. The changes in crystallin expression and fibre differentiation were delayed, but cultures of the faster growing genotypes were least affected. With further culture, crystallin expression tended to recover to control values although levels of fibre differentiation and cell growth remained depressed. The results indicate that genetic differences in intrinsic growth rate moderate but do not change the programme of crystallin expression shown by lens epithelial cells in culture, and that this programme shows resistance to change.

Alterations in crystallin gene expression during subculture of chick lens cells.

We report here on the changes in crystallin gene expression during serial subculture of lens epithelial cells derived from day-old post-hatch chicks. Total cellular RNA from mass cultures were analysed by in vitro cell-free translation and by RNA blot (Northern) hybridization using a cloned delta-crystallin cDNA. Our results indicate that following subculture, lens epithelial cells which still retain the capacity for lens fibre differentiation (lentoid body formation) show a selective loss of delta-crystallin synthesis, and that this is related to the loss of delta-crystallin mRNA. The data suggest that older epithelial-cell populations give rise to lentoid bodies which in terms of crystallin gene expression closely resemble the later-formed cortical fibres of the adult chick lens. Tertiary cultures had an accelerated growth rate, formed no lentoids, contained no translatable alpha- or delta-crystallin mRNAs but still contained translatable beta-crystallin mRNAs.

Developmental changes in membrane protein expression by chick lens cells in vivo and in vitro and the detection of main intrinsic polypeptide (MIP).

We have compared the long-term developmental changes in water-insoluble protein expression by chick lens cells in vitro and in vivo. Crude membrane fractions were prepared by alkali treatment of the urea-insoluble protein fraction, and the proteins analysed by sodium dodecyl sulphate-polyacrylamide (SDS-PAGE) gel electrophoresis. The major component present in the urea-insoluble fraction of chick lens fibres, a 25,000 MW polypeptide (MIP-25K) was more abundant in adult (8 weeks) than day-old post-hatch chick lens fibre masses. MIP-25K was detected in differentiated but not predifferentiated lens cell cultures, and indirect immunofluorescence using anti-bovine MIP antiserum indicated that MIP-25K was localized in the lentoid bodies. Our findings indicate that the urea-insoluble protein profiles of long-term well-differentiated chick lens cell cultures are qualitatively very similar to the profiles of the lens fibres. The data also confirm that the expression of MIP-25K, rather than the expression of water-soluble crystallin protein, is a marker for lens cell differentiation, and confirm earlier reports, which have been disputed, that delta-crystallin (but not alpha-or beta-crystallin) is specifically associated with chick lens fibre membranes.

A comparison of the changing patterns of crystallin expression in vivo, in long-term primary cultures in vitro and in response to a carcinogen.

Changes in the differentiation of day-old chick lens epithelium in long-term primary culture conditions were investigated by sodium dodecyl sulphate-polyacrylamide electrophoresis, using integrating densitometry to assess the relative levels of accumulated crystallin and non-crystallin polypeptides and fluorography to assess their relative levels of synthesis. The main changes during the culture period included a relative decline in the proportion of actin and other non-crystallins, an initial increase in 48K delta-crystallin expression followed by a decline and a shift in beta-crystallin expression from a relative preponderance of the 24K and 23K polypeptides to a relative preponderance of the 24K and 22K polypeptides. At all stages the level of the 19K alpha-crystallin was higher than that of the 20K alpha-crystallin polypeptide. In general, the changes in the pattern of expression of these polypeptides in culture were similar to those observed in vivo in the post-hatch chick, suggesting an intrinsic programme of crystallin expression. The changes in gene expression were also tested indirectly by brief exposure of the cells in vitro to a carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) which is known to produce, in some systems, effects related to the status of the cell at the time of treatment. The effects were found to depend on the stage of differentiation of the culture at the time of treatment. Treatment on day 1 of culture prevented later lentoid formation and severely reduced the expression of all crystallins with the exception of the 34K beta-crystallin polypeptide. Actin was the most abundant soluble cell component, and a proportion of the cells acquired a fibroblast-like morphology. Treatment with MNNG on day 7 led to a delay in lentoid formation and a differential reduction of the synthesis of crystallin polypeptides, whereas the treatment of already differentiated cultures on day 18 and to lesser extents on days 27, 45 and 55, respectively, led to an increase in crystallin synthesis relative to controls. These results suggest that this programme of crystallin gene expression becomes more resistant to change with increasing epithelial differentiation.

Factors affecting steroid and prostaglandin secretion by reproductive tissues of cycling and pregnant sows in vitro.

The secretion of prostaglandin E2 and prostaglandin F2 alpha by endometrial and corpus luteum tissues of cycling and pregnant sows in vitro has been measured and the effects of arachidonic acid and steroids on prostaglandin secretion studied. In general, both types of tissues in vitro exhibited a gradually increasing capacity to secrete prostaglandin as the cycle progressed, although the most significant increase was in prostaglandin F2 alpha secretion. An inverse relationship between prostaglandin and progesterone secretion was observed, and, while indomethacin inhibited prostaglandin secretion, it was without effect on progesterone. A much lower capacity for prostaglandin secretion was observed with tissues from pregnant sows. Prostaglandin secretion by endometrial and corpus luteum tissues of the cycle were stimulated by exogenous arachidonic acid which also stimulated prostaglandin E2 secretion in pregnant sows. This clearly suggests that phospholipase A2 activity which controls substrate availability is of considerable importance in pig reproductive tissues. The effects of steroid hormones on prostaglandin secretion by pig reproductive tissues was more complex although in general oestradiol appeared to be inhibitory while progesterone was stimulatory.