Replacement of K-Ras with H-Ras supports normal embryonic development despite inducing cardiovascular pathology in adult mice

Ras proteins are highly related GTPases that have key roles in regulating growth, differentiation and tumorigenesis. Gene-targeting experiments have shown that, out of the three mammalian ras genes, only K-ras is essential for normal mouse embryogenesis, and that mice deprived of H-ras and/or N-ras show no major phenotype. We generated mice (HrasKI) in which the K-ras gene had been modified to encode H-Ras protein. HrasKI mice produce undetectable amounts of K-Ras but-in contrast to mice homozygous for a null K-ras allele-they are born at the expected mendelian frequency, indicating that H-Ras can be substituted for K-Ras in embryonic development. However, adult HrasKI mice show dilated cardiomyopathy associated with arterial hypertension. Our results show that K-Ras can be replaced by H-Ras in its essential function in embryogenesis, and indicate that K-Ras has a unique role in cardiovascular homeostasis.     

Involvement of H- and N-Ras isoforms in transforming growth factor-beta1-induced proliferation and in collagen and fibronectin synthesis

Transforming growth factor beta1 (TGF-beta1) has a relevant role in the origin and maintenance of glomerulosclerosis and tubule-interstitial fibrosis. TGF-beta and Ras signaling pathways are closely related: TGF-beta1 overcomes Ras mitogenic effects and Ras counteracts TGF-beta signaling. Tubule-interstitial fibrosis is associated to increases in Ras, Erk, and Akt activation in a renal fibrosis model. We study the role of N- and H-Ras isoforms, and the involvement of the Ras effectors Erk and Akt, in TGF-beta1-mediated extracellular matrix (ECM) synthesis and proliferation, using embrionary fibroblasts from double knockout (KO) mice for H- and N-Ras (H-ras(-/-)/N-ras(-/-)) isoforms and from heterozygote mice (H-ras(+/-)/N-ras(+/-)). ECM synthesis is increased in basal conditions in H-ras(-/-)/N-ras(-/-) fibroblasts, this increase being higher after stimulation with TGF-beta1. TGF-beta1-induced fibroblast proliferation is smaller in H-ras(-/-)/N-ras(-/-) than in H-ras(+/-)/N-ras(+/-) fibroblasts. Erk activation is decreased in H-ras(-/-)/N-ras(-/-) fibroblasts; inhibition of Erk activation reduces fibroblast proliferation. Akt activation is higher in double KO fibroblasts than in heterozygotes; inhibition of Akt activation also inhibits ECM synthesis. We suggest that H- and N-Ras isoforms downregulate ECM synthesis, and mediate proliferation, in part through MEK/Erk activation. PI3K-Akt pathway activation may be involved in the increase in ECM synthesis observed in the absence of H- and N-Ras.     

Targeted disruption of Ras-Grf2 shows its dispensability for mouse growth and development

The mammalian Grf1 and Grf2 proteins are Ras guanine nucleotide exchange factors (GEFs) sharing a high degree of structural homology, as well as an elevated expression level in central nervous system tissues. Such similarities raise questions concerning the specificity and/or redundancy at the functional level between the two Grf proteins. grf1-null mutant mice have been recently described which showed phenotypic growth reduction and long-term memory loss. To gain insight into the in vivo function of Grf2, we disrupted its catalytic CDC25-H domain by means of gene targeting. Breeding among grf2(+/-) animals gave rise to viable grf2(-/-) adult animals with a normal Mendelian pattern, suggesting that Grf2 is not essential for embryonic and adult mouse development. In contrast to Grf1-null mice, analysis of grf2(-/-) litters showed similar size and weight as their heterozygous or wild-type grf2 counterparts. Furthermore, adult grf2(-/-) animals reached sexual maturity at the same age as their wild-type littermates and showed similar fertility levels. No specific pathology was observed in adult Grf2-null animals, and histopathological studies showed no observable differences between null mutant and wild-type Grf2 mice. These results indicate that grf2 is dispensable for mouse growth, development, and fertility. Furthermore, analysis of double grf1/grf2 null animals did not show any observable phenotypic difference with single grf1(-/-) animals, further indicating a lack of functional overlapping between the two otherwise highly homologous Grf1 and Grf2 proteins.     

Structure and activation of the human N-ras gene

The normal human N-ras gene has been cloned. In structure and sequence it closely resembles the human H-ras and K-ras genes. The three genes share regions of nucleotide homology and nucleotide divergence within coding sequences and have a common intron/exon structure, indicating that they have evolved from a similarly spliced ancestral gene. The N-ras gene of SK-N-SH neuroblastoma cells has transforming activity, while the normal N-ras gene does not, the result of a single nucleotide change substituting lysine for glutamine in position 61 of the N-ras gene product. From previous studies we conclude that amino acid substitutions in two distinct regions can activate the transforming potential of ras gene products.     

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(tmDelta4A/+) mice produced viable K-ras(tmDelta4A/tmDelta4A) offspring with the expected Mendelian ratios of inheritance, and these mice expressed the K-ras 4B splice variant only. K-ras(tmDelta4A/tmDelta4A) mice were fertile and showed no histopathological abnormalities on inbred (129/Ola) or crossbred (129/Ola x 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.     

Ras-guanine nucleotide exchange factor sos2 is dispensable for mouse growth and development

The mammalian sos1 and sos2 genes encode highly homologous members of the Son-of-sevenless family of guanine nucleotide exchange factors. They are ubiquitously expressed and play key roles in transmission of signals initiated by surface protein tyrosine kinases that are transduced into the cell through the action of membrane-associated Ras proteins. Recent reports showed that targeted disruption of the sos1 locus results in embryonic lethality. To gain insight into the in vivo function of sos2, we disrupted its catalytic CDC25-H domain by means of gene targeting techniques. Mating among heterozygous sos2(+/-) mice produced viable sos2(-/-) offspring with a normal Mendelian pattern of inheritance, indicating that the loss of sos2 does not interfere with embryo viability in the uterus. Adult homozygous mutant sos2(-/-) mice reached sexual maturity at the same age as their wild-type littermates, and both male and female null mutants were fertile. Histopathological analysis showed no observable differences between mutant and wild-type mice. Our results show that unlike the case for sos1, sos2 gene function is dispensable for normal mouse development, growth, and fertility.     

甲醛致大鼠鼻腔癌与K-ras癌基因活化相关

为探讨甲醛致大鼠鼻腔癌的分子机制,对甲醛诱发的大鼠鼻腔癌细胞系FAT7中的转化序列进行检测.采用的实验方法,包括肿瘤细胞DNA与选择标志基因(neo)共转染、裸鼠成瘤性分析、Southern杂交、聚合酶链反应(PCR)和序列分析等.结果发现:在第二轮裸鼠肿瘤DNA中含有大鼠源性的K-ras基因序列,而无大鼠源性的H-ras、N-ras和p53基因序列.这表明甲醛诱发的大鼠鼻腔癌细胞系FAT7中所含的转化序列与H-ras、N-ras及p53基因无关,K-ras癌基因的活化可能参与甲醛致大鼠鼻腔癌.     

Oncogenicity of human N-ras oncogene and proto-oncogene introduced into retroviral vectors.

The N-ras gene is the only member of the ras family which has never been naturally transduced into a retrovirus. In order to study the in vitro and in vivo oncogenicity of N-ras and to compare its pathogenicity to that of H-ras, we have inserted an activated or a normal form of human N-ras cDNA into a slightly modified Harvey murine sarcoma virus-derived vector in which the H-ras p21 coding region had been deleted. The resulting constructions were transfected into NIH 3T3 cells. The activated N-ras-containing construct (HSN) induced 10(4) foci per microgram of DNA and was found to be as transforming as H-ras was. After infection of the transfected cells by either the ecotropic Moloney murine leukemia virus or the amphotropic 4070A helper viruses, rescued transforming viruses were injected into newborn mice. Both pseudotypes of HSN virus containing activated N-ras induced the typical Harvey disease with similar latency. However, we found that the virus which contained normal N-ras p21 (HSn) was also pathogenic and induced splenomegaly, lymphadenopathies, and sarcoma in mice after a latency of 3 to 7 weeks. In addition, Moloney murine leukemia virus pseudotypes of N-ras caused neurological disorders in 30% of the infected animals. These results differed markedly from those of previous experiments in which we had inserted the activated form of N-ras in the pSV(X) vector: the resulting SVN-ras virus was transforming on NIH 3T3 cells but was poorly oncogenic in vivo (M. Souyri, C. F. Koehne, P. V. O'Donnel, T. H. Aldrich, M. E. Furth, and E. Fleissner, Virology 158:69-78). However, similarly poor oncogenicity was also observed when the v-H-ras coding sequence was inserted in pSV(X) vector, which indicated that the vector sequences play a crucial role in the pathogenicity of a given oncogene. Altogether, these data demonstrated unequivocally that N-ras is potentially as oncogenic as H-ras and that such oncogenic effect could depend on the vector environment.     

Defective mammopoiesis, but normal hematopoiesis, in mice with a targeted disruption of the prolactin gene.

Prolactin (PRL) has been implicated in numerous physiological and developmental processes. The mouse PRL gene was disrupted by homologous recombination. The mutation caused infertility in female mice, but did not prevent female mice from manifesting spontaneous maternal behaviors. PRL-deficient males were fertile and produced offspring with normal Mendelian gender and genotype ratios when they were mated with heterozygous females. Mammary glands of mutant female mice developed a normal ductal tree, but the ducts failed to develop lobular decorations, which is a characteristic of the normal virgin adult mammary gland. The potential effect of PRL gene disruption on antigen-independent primary hematopoiesis was assessed. The results of this analysis indicated that myelopoiesis and primary lymphopoiesis were unaltered in the mutant mice. Consistent with these observations in PRL mutant mice, PRL failed to correct the bone marrow B cell deficiency of Snell dwarf mice. These results argue that PRL does not play any indispensable role in primary lymphocyte development and homeostasis, or in myeloid differentiation. The PRL-/- mouse model provides a new research tool with which to resolve a variety of questions regarding the involvement of both endocrine and paracrine sources of PRL in reproduction, lactogenesis, tumorigenesis and immunoregulation.     

Point mutations of ras oncogenes are an early event in thyroid tumorigenesis

Identifying the nature of the genetic mutations in thyroid neoplasms and their prevalence in the various tumor phenotypes is critical to understanding their pathogenesis. Mutational activation of ras oncogenes in human tumors occurs predominantly through point mutations in two functional regions of the molecules, codons 12, 13 (GTP-binding domain) or codon 61 (GTPase domain). We examined the prevalence of point mutations in codons 12, 13, and 61 of the oncogenes K-ras, N-ras, and H-ras in benign and malignant human thyroid tumors by hybridization of PCR-amplified tumor DNA with synthetic oligodeoxynucleotide probes. None of the eight normal thyroid tissues harbored point mutations. Four of nineteen nodules from multinodular goiters (21%), 6/24 microfollicular adenomas (25%), 3/14 papillary carcinomas (21%), and 0/3 follicular carcinomas contained ras point mutations. The predominant mutation was a valine for glycine substitution in codon 12 of H-ras. None of the multinodular goiter tumors known to be polyclonal (and thus due to hyperplasia) had point mutations, whereas one of the two monoclonal adenomas arising in nodular glands contained in H-ras codon 12 valine substitution, which was confirmed by sequencing the tumor DNA. These data show that ras activation is about equally prevalent in benign and malignant thyroid neoplasms, and thus may be an early event in the tumorigenic process.     

A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane

The C-terminal CAAX motif of ras proteins undergoes a triplet of posttranslational modifications that are required for membrane association. The CAAX motif lies immediately C-terminal to the hypervariable domain, a region of 20 amino acids that distinguishes the ras proteins from each other. The hypervariable domains of p21H-ras, p21N-ras, and p21K-ras(A) contain sites for palmitoylation, which we now show must combine with the CAAX motif to target specific plasma membrane localization. Within the hypervariable domain of p21K-ras(B), which is not palmitoylated, we have identified a novel plasma membrane targeting signal consisting of a polybasic domain that also acts in combination with the CAAX motif. One function of the hypervariable domains of p21ras is therefore to provide different signals for plasma membrane localization.     

Reduced sensitivity to and ras mutation spectrum of N-ethyl-N-nitrosourea-induced thymic lymphomas in adult C.B-17 scid mice

Scid mice are defective in the ability to repair DNA double strand breaks and, as a consequence, their cells are radiosensitive. Further, they have been shown to be prone to develop thymic lymphomas (TLs) after small doses of ionizing radiation. Little is known, however, on the role of scid mutation in chemical carcinogenesis. To determine if scid mutation increased predisposition to chemical carcinogenesis, we examined both the susceptibility of scid mice to N-ethyl-N-nitrosourea (ENU)-induced lymphomagenesis and the involvement of ras gene activation. Adult female mice at 8 weeks of age were given ENU in their drinking water at 400 ppm for 2-10 weeks. Contrary to expectations, we observed a two to three-fold reduction in TL development in the scid mice. The highest incidence was achieved by ENU treatment for 8 weeks for scid and wild-type C.B-17 mice, of 42 and 85%, respectively (P<0.05). We investigated whether this was attributable to the usage of the ras mutation pathway. There was, however, no significant difference in the frequency and spectrum of K-ras mutation between the scid and wild-type C.B-17 mice. Most of the K-ras mutations were either GGT to GAT transition in codon 12 (11/23: 48%) or CAA to CCA transversion in codon 61 (8/23: 35%) that was independent of scid background. The incidence of N-ras mutation was very low. These results indicate that scid mice are less susceptible to ENU-induced lymphomagenesis and ras gene mutation frequently occurs in both scid and wild-type C.B-17 mice.     

Snm1-deficient mice exhibit accelerated tumorigenesis and susceptibility to infection

The eukaryotic SNM1 gene family has been implicated in a number of cellular pathways, including repair of DNA interstrand cross-links, involvement in VDJ recombination, repair of DNA double-strand breaks, and participation in cell cycle checkpoint pathways. In particular, mammalian SNM1 has been shown to be required in a mitotic checkpoint that causes arrest of cells in prophase prior to chromosome condensation in response to spindle poisons. Here, we report on the phenotype of a knockout of Snm1 in the mouse. Snm1-/- mice are viable and fertile but exhibit a complex phenotype. Both homozygous and heterozygous mice show a decline in survival compared to wild-type littermates. In homozygous mutant males, this reduction in survival is principally due to bacterial infections in the preputial and mandibular glands and to a lesser extent to tumorigenesis, while in homozygous and heterozygous females, it is due almost solely to tumorigenesis. The high incidence of bacterial infections in the homozygous mutant males suggests an immune dysfunction; however, examinations of T- and B-cell development and immunoglobulin class switching did not reveal a defect in these pathways. Crossing of Snm1 mutant mice with a Trp53 null mutant resulted in an increase in mortality and a restriction of the tumor type to lymphomas, particularly those of the thymus. Taken together, these findings demonstrate that Snm1 is a tumor suppressor in mice that in addition has a role in immunity.     

Decreased viability of nitric oxide synthase double knockout mice

Nitric oxide acts as an important intracellular messenger in a variety of systems, including reproduction. Previous studies have shown the importance of nitric oxide in embryo development. NO is produced from l-arginine by the enzyme, nitric oxide synthase (NOS), which has three isoforms: endothelial (NOS3), neural (NOS1), and inducible (NOS2). We hypothesize that, because of the importance of NOS in development, at least two NOS isoforms are required in order for normal embryo development to occur. Through the generation of NOS3/NOS2, NOS3/NOS1, and NOS2/NOS1 double knockout mice, we found that while litter size remains unchanged, the expected number of generated double knockout mice varies significantly from what would be predicted by Mendelian genetics. Estrous cycles were similar for both DKO and the wild-type mice, and both groups were deemed fertile by their ability to mate with wild-type (CD-1) mice. Together, these results lead us to conclude that the lack of two NOS isoforms leads to a decreased viability in mice because of a developmental problem in the double knockout embryo.     

Loss of the Atp2c1 secretory pathway Ca(2+)-ATPase (SPCA1) in mice causes Golgi stress, apoptosis, and midgestational death in homozygous embryos and squamous cell tumors in adult heterozygotes

Loss of one copy of the human ATP2C1 gene, encoding SPCA1 (secretory pathway Ca(2+)-ATPase isoform 1), causes Hailey-Hailey disease, a skin disorder. We performed targeted mutagenesis of the Atp2c1 gene in mice to analyze the functions of this Golgi membrane Ca(2+) pump. Breeding of heterozygous mutants yielded a normal Mendelian ratio among embryos on gestation day 9.5; however, null mutant (Spca1(-/-)) embryos exhibited growth retardation and did not survive beyond gestation day 10.5. Spca1(-/-) embryos had an open rostral neural tube, but hematopoiesis and cardiovascular development were ostensibly normal. Golgi membranes of Spca1(-/-) embryos were dilated, had fewer stacked leaflets, and were expanded in amount, consistent with increased Golgi biogenesis. The number of Golgi-associated vesicles was also increased, and rough endoplasmic reticulum had fewer ribosomes. Coated pits, junctional complexes, desmosomes, and basement membranes appeared normal in mutant embryos, indicating that processing and trafficking of proteins in the secretory pathway was not massively impaired. However, apoptosis was increased, possibly the result of secretory pathway stress, and a large increase in cytoplasmic lipid was observed in mutant embryos, consistent with impaired handling of lipid by the Golgi. Adult heterozygous mice appeared normal and exhibited no evidence of Hailey-Hailey disease; however, aged heterozygotes had an increased incidence of squamous cell tumors of keratinized epithelial cells of the skin and esophagus. These data show that loss of the Golgi Ca(2+) pump causes Golgi stress, expansion of the Golgi, increased apoptosis, and embryonic lethality and demonstrates that SPCA1 haploinsufficiency causes a genetic predisposition to cancer.     

The farnesyl transferase inhibitor SCH 66336 induces a G(2) --> M or G(1) pause in sensitive human tumor cell lines

SCH 66336 is a potent farnesyl transferase inhibitor (FTI) in clinical development. It efficiently prevents the membrane association of H-ras, but not K- or N-ras. Yet, in soft agar, it reverts the anchorage-independent growth of human tumor cell lines (hTCLs) harboring H-ras, K-ras, and N-ras mutations, implying that blocking farnesylation of proteins besides ras may be responsible for this effect. Experiments show that SCH 66336 altered the cell cycle distribution of sensitive human tumor cells in two distinct ways. Most sensitive hTCLs accumulated in the G(2)-->M phase after the FTI treatment, but those with an activated H-ras accumulated in G(1) phase, suggesting that the biological effects induced by FTIs in cells with an activated H-ras are distinct from other sensitive cells. A careful genotypic comparison of the hTCLs revealed that those cells with wild-type p53 are especially sensitive to the FTIs. In these cells p53 and its downstream target gene p21(Cip1) are induced after treatment with SCH 66336 for 24 h. These data suggest that cell cycle effects, either G(1) or G(2)-->M accumulation, and p53 status are important for mediating the effects of FTIs on tumor cells.