Autocrine stimulation of intracellular PDGF receptors in v-sis-transformed cells

Autocrine activation of platelet-derived growth factor (PDGF) receptors is the mechanism of transformation by the v-sis oncogene. Since the addition of PDGF does not transform normal cells, autocrine mechanisms may involve unique pathways of receptor activation. In this study autocrine stimulation of the PDGF receptor was observed in v-sis-transformed normal rat kidney (NRK) cells. In contrast to receptor activation in normal cells, autocrine activation of PDGF receptors in v-sis-transformed cells occurred in intracellular compartments, disrupting receptor processing and diverting receptors and their precursors to a chloroquine-sensitive degradation pathway. These findings show that intracellular activation of receptors by autocrine mechanisms may play a role in cell transformation.     

Evidence that the v-sis gene product transforms by interaction with the receptor for platelet-derived growth factor

A scheme for partial purification of biologically active v-sis-coded protein from cells transformed with simian sarcoma virus (SSV) has made possible a functional comparison of the transforming protein with platelet-derived growth factor (PDGF). The SSV-transforming gene product is capable of specifically binding PDGF receptors, stimulating tyrosine phosphorylation of PDGF receptors, and inducing DNA synthesis in quiescent fibroblasts. Each of these activities was specifically inhibited by antibodies to different regions of the v-sis gene product. Moreover, viral infection of a variety of cell types revealed a strict correlation between those cells possessing PDGF receptors and those susceptible to transformation by SSV. These findings provide evidence that SSV-transforming activity is mediated by the interaction of a virus-coded mitogen with PDGF receptors.     

The role of individual cysteine residues in the structure and function of the v-sis gene product

The v-sis oncogene encodes a platelet-derived growth factor (PDGF)-related product whose transforming activity is mediated by its functional interaction with the PDGF receptor. PDGF, as well as processed forms of the v-sis gene product, is a disulfide-linked dimer with eight conserved cysteine residues in the minimum region necessary for biologic activity. Site-directed mutagenesis of the v-sis gene revealed that each conserved cysteine residue was required directly or indirectly for disulfide-linked dimer formation. However, substitution of serine for cysteine codons at any of four positions had no detrimental effect on transforming activity of the encoded v-sis protein. These results establish that interchain disulfide bonds are not essential in order for this protein to act as a functional ligand for the PDGF receptor. The remaining four substitutions of serine for cysteine each inactivated transforming function of the molecule. In each case this was associated with loss of a conformation shown to involve intramolecular disulfide bonds. These studies provide insight into the role of individual cysteine residues in determining the structure of the sis/PDGF molecule critical for biological activity.     

Suppression of human colorectal carcinoma cell growth by wild-type p53

Mutations of the p53 gene occur commonly in colorectal carcinomas and the wild-type p53 allele is often concomitantly deleted. These findings suggest that the wild-type gene may act as a suppressor of colorectal carcinoma cell growth. To test this hypothesis, wild-type or mutant human p53 genes were transfected into human colorectal carcinoma cell lines. Cells transfected with the wild-type gene formed colonies five- to tenfold less efficiently than those transfected with a mutant p53 gene. In those colonies that did form after wild-type gene transfection, the p53 sequences were found to be deleted or rearranged, or both, and no exogenous p53 messenger RNA expression was observed. In contrast, transfection with the wild-type gene had no apparent effect on the growth of epithelial cells derived from a benign colorectal tumor that had only wild-type p53 alleles. Immunocytochemical techniques demonstrated that carcinoma cells expressing the wild-type gene did not progress through the cell cycle, as evidenced by their failure to incorporate thymidine into DNA. These studies show that the wild-type gene can specifically suppress the growth of human colorectal carcinoma cells in vitro and that an in vivo-derived mutation resulting in a single conservative amino acid substitution in the p53 gene product abrogates this suppressive ability.     

Expression of a platelet-derived growth factor-like protein in simian sarcoma virus transformed cells

The near identity of the partial amino acid sequence of human platelet-derived growth factor (PDGF) and that predicted for p28sis, the putative transforming protein of the simian sarcoma virus (SSV), suggests expression of a growth factor activity may be central for transformation by SSV. It is now reported that SSV-transformed cells but not control cells contain a growth factor activity that is identical to PDGF in immunoassay, in mitogenic dose response, and in specific mitogenic activity. The protein immunoprecipitated by antiserum to human PDGF has an apparent molecular weight of 20,000, identical to that of p20sis, the putative intracellular degradation product of p28sis. The results support the concept that expression of a PDGF-like molecule, which appears to be the product of the viral-sis gene, is responsible for the abnormal regulation of growth is SSV-transformed cells.     

Molecular localization of the transforming and secretory properties of PDGF A and PDGF B

Human platelet-derived growth factor (PDGF) is a connective tissue cell mitogen comprised of two related chains encoded by distinct genes. The B chain is the homolog of the v-sis oncogene product. Properties that distinguish these ligands include greater transforming potency of the B chain and more efficient secretion of the A chain. By a strategy involving the generation of PDGF A and B chimeras, these properties were mapped to distinct domains of the respective molecules. Increased transforming efficiency segregated with the ability to activate both alpha and beta PDGF receptors. These findings genetically map PDGF B residues 105 to 144 as responsible for conformational alterations critical to beta PDGF receptor interaction and provide a mechanistic basis for the greater transforming potency of the PDGF B chain.     

Role of phosphatidylinositol kinase in PDGF receptor signal transduction

The molecules with which the platelet-derived growth factor (PDGF) receptor interacts to elicit the biochemical reactions responsible for cell proliferation have not been identified. Antisera directed against specific PDGF receptor peptides coprecipitated a phosphatidylinositol (PI) kinase and the PDGF receptor. Immunoprecipitates from PDGF-stimulated cells contained 10 to 50 times as much PI kinase as those from unstimulated cells. Mutation of the PDGF receptor by deletion of its kinase insert region resulted in a receptor markedly less effective than the wild type in eliciting cell proliferation and defective in PDGF-stimulated PI kinase, but still capable of PDGF-induced receptor autophosphorylation and phosphoinositide hydrolysis. These data show that the PDGF receptor is physically associated with a PDGF-sensitive PI kinase that is distinct from tyrosine kinase and is not required for PDGF-induced PI hydrolysis. The finding that the mutant PDGF receptor missing the kinase insert domain elicited known early biochemical responses to PDGF, but did not associate with or regulate PI kinase, suggests a novel role for the receptor-associated PI kinase in the transmission of mitogenic signals.     

HSP104 required for induced thermotolerance

A heat shock protein gene, HSP104, was isolated from Saccharomyces cerevisiae and a deletion mutation was introduced into yeast cells. Mutant cells grew at the same rate as wild-type cells and died at the same rate when exposed directly to high temperatures. However, when given a mild pre-heat treatment, the mutant cells did not acquire tolerance to heat, as did wild-type cells. Transformation with the wild-type gene rescued the defect of mutant cells. The results demonstrate that a particular heat shock protein plays a critical role in cell survival at extreme temperatures.     

Simian sarcoma virus--transformed cells secrete a mitogen identical to platelet-derived growth factor

Normal rat kidney (NRK) cells transformed by simian sarcoma virus (SSV) release into the culture medium a biologically active mitogen with properties identical to those of human platelet-derived growth factor (PDGF). Like PDGF, the growth factor derived from SSV-NRK cells was shown to be stable to heat and sensitive to reducing agents. It was capable of inhibiting binding of labeled PDGF to the receptor on human fibroblasts. It also stimulated the phosphorylation of the same membrane protein (185 kilodaltons) in isolated plasma membranes from human fibroblasts. Immunoprecipitation of metabolically labeled proteins released by SSV-NRK cells showed that a 34-kilodalton protein was specifically precipitated by antiserum to PDGF. Upon reduction, this protein had a molecular size of 17 kilodaltons. PDGF has been shown to consist of two 14- to 18-kilodalton proteins linked by disulfide bonds.     

ras-transformed cells: altered levels of phosphatidylinositol-4,5-bisphosphate and catabolites

Steady-state cellular levels of phosphatidylinositol-4,5-bisphosphate (PIP2), 1,2-diacylglycerol (DAG), and inositol phosphates have been measured in two different fibroblast cell lines (NIH 3T3 and NRK cells) before and after transformation with three different ras genes. At high cell density the ratio of DAG to PIP2 was 2.5- to 3-fold higher in the ras-transformed cells than in their untransformed counterparts. The sum of the water-soluble breakdown products of the polyphosphoinositides, inositol-1,4-bisphosphate and inositol-1,4,5-trisphosphate, was also elevated in ras-transformed NRK cells compared with nontransformed NRK cells. These findings suggest that the ras (p21) protein may act by affecting these levels, possibly as a regulatory element in the PIP2 breakdown pathway.     

Comparison of biological properties and transforming potential of human PDGF-A and PDGF-B chains

Human platelet-derived growth factor (PDGF) consists of two distinct but related polypeptide chains designated PDGF-A and PDGF-B. The gene encoding PDGF-B has given rise to the v-sis oncogene. In the present study the transforming activities of PDGF-A and PDGF-B genes are compared. The PDGF-A chain gene is markedly less efficient in inducing transformation than the PDGF-B gene under the influence of the same promoter. There are significant differences in the secretory and growth stimulating properties of the two chains. These properties appear to account for the much more potent transforming ability of the PDGF-B gene. These findings provide insights into biologic properties of a growth factor responsible for potent autocrine stimulation of abnormal cell proliferation.     

Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms

Familial cancer syndromes have helped to define the role of tumor suppressor genes in the development of cancer. The dominantly inherited Li-Fraumeni syndrome (LFS) is of particular interest because of the diversity of childhood and adult tumors that occur in affected individuals. The rarity and high mortality of LFS precluded formal linkage analysis. The alternative approach was to select the most plausible candidate gene. The tumor suppressor gene, p53, was studied because of previous indications that this gene is inactivated in the sporadic (nonfamilial) forms of most cancers that are associated with LFS. Germ line p53 mutations have been detected in all five LFS families analyzed. These mutations do not produce amounts of mutant p53 protein expected to exert a trans-dominant loss of function effect on wild-type p53 protein. The frequency of germ line p53 mutations can now be examined in additional families with LFS, and in other cancer patients and families with clinical features that might be attributed to the mutation.     

Prevention of Brca1-mediated mammary tumorigenesis in mice by a progesterone antagonist

Women with mutations in the breast cancer susceptibility gene BRCA1 are predisposed to breast and ovarian cancers. Why the BRCA1 protein suppresses tumor development specifically in ovarian hormone-sensitive tissues remains unclear. We demonstrate that mammary glands of nulliparous Brca1/p53-deficient mice accumulate lateral branches and undergo extensive alveologenesis, a phenotype that occurs only during pregnancy in wild-type mice. Progesterone receptors, but not estrogen receptors, are overexpressed in the mutant mammary epithelial cells because of a defect in their degradation by the proteasome pathway. Treatment of Brca1/p53-deficient mice with the progesterone antagonist mifepristone (RU 486) prevented mammary tumorigenesis. These findings reveal a tissue-specific function for the BRCA1 protein and raise the possibility that antiprogesterone treatment may be useful for breast cancer prevention in individuals with BRCA1 mutations.     

Genetic mechanisms of tumor suppression by the human p53 gene

Mutations of the gene encoding p53, a 53-kilodalton cellular protein, are found frequently in human tumor cells, suggesting a crucial role for this gene in human oncogenesis. To model the stepwise mutation or loss of both p53 alleles during tumorigenesis, a human osteosarcoma cell line, Saos-2, was used that completely lacked endogenous p53. Single copies of exogenous p53 genes were then introduced by infecting cells with recombinant retroviruses containing either point-mutated or wild-type versions of the p53 cDNA sequence. Expression of wild-type p53 suppressed the neoplastic phenotype of Saos-2 cells, whereas expression of mutated p53 conferred a limited growth advantage to cells in the absence of wild-type p53. Wild-type p53 was phenotypically dominant to mutated p53 in a two-allele configuration. These results suggest that, as with the retinoblastoma gene, mutation of both alleles of the p53 gene is essential for its role in oncogenesis.     

Inhibition of PDGF beta receptor signal transduction by coexpression of a truncated receptor.

A mutated form of the platelet-derived growth factor (PDGF) beta receptor lacking most of its cytoplasmic domain was tested for its ability to block wild-type PDGF receptor function. PDGF induced the formation of complexes consisting of wild-type and truncated receptors. Such complexes were defective in autophosphorylation. When truncated receptors were expressed in excess compared to wild-type receptors, stimulation by PDGF of receptor autophosphorylation, association of phosphatidylinositol-3 kinase with the receptor, and calcium mobilization were blocked. Thus, a truncated receptor can inactivate wild-type receptor function by forming ligand-dependent receptor complexes (probably heterodimers) that are incapable of mediating the early steps of signal transduction.     

Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations

Heterozygous mutations encoding abnormal forms of the death receptor Fas dominantly interfere with Fas-induced lymphocyte apoptosis in human autoimmune lymphoproliferative syndrome. This effect, rather than depending on ligand-induced receptor oligomerization, was found to stem from ligand- independent interaction of wild-type and mutant Fas receptors through a specific region in the extracellular domain. Preassociated Fas complexes were found in living cells by means of fluorescence resonance energy transfer between variants of green fluorescent protein. These results show that formation of preassociated receptor complexes is necessary for Fas signaling and dominant interference in human disease.     

Binding of GAP to activated PDGF receptors

The ras proto-oncogene products appear to relay intracellular signals via the Ras guanosine triphosphatase (GTPase) activator protein, GAP. In dog epithelial cells expressing human platelet-derived growth factor (PDGF) receptors, binding of PDGF caused approximately one-tenth of the total GAP molecules to complex with the receptor. Studies with mutant PDGF receptors showed that maximum association required both receptor kinase activity and phosphorylatable tyrosine residues at both the identified sites of receptor autophosphorylation.     

通过缺失大丽轮枝菌Vdku80构建其高效基因敲除受体菌株

【目的】验证大丽轮枝菌（Verticillium dahliae）非同源末端连接途径关键基因Vdku80的功能,构建高效的大丽轮枝菌基因敲除受体菌株。【方法】利用常规基因敲除的方法,构建大丽轮枝菌Vdku80基因缺失的突变体菌株ΔVdku80。即将Vdku80的同源重组DNA片段（Vdku80的基因组上下游DNA片段之间连接潮霉素抗性基因）通过农杆菌介导的转化转入大丽轮枝菌,通过同源区段的重组,潮霉素抗性基因便会替换掉野生型Vdku80,从而获得突变体菌株ΔVdku80。对突变体菌株ΔVdku80的生物学表型,即生长速率、产孢量和对棉花的致病性进行测试。分别以大丽轮枝菌野生型菌株和ΔVdku80作为受体菌株,采用上述基因敲除方法测试2个几丁质合成酶编码基因ChsV和ChsVI的基因敲除效率。【结果】成功构建了Vdku80缺失突变体ΔVdku80。野生型和突变体菌株ΔVdku80在PDA培养基上的菌落形态相似,且培养8 d后菌落直径无明显差别。野生型和突变体菌株ΔVdku80产孢高峰均出现在摇瓶培养后第5天,且该时期产孢量亦无明显的变化。浓度为0.02%的MMS对野生型和突变体菌株ΔVdku80的生长均有明显的抑制作用,且抑制程度相同。接种野生型和突变体菌株ΔVdku80 4周后,棉株真叶均开始表现出萎蔫、褪绿等发病症状,野生型和突变体菌株ΔVdku80对棉花造成的病情指数分别为54.2±4.9和53.6±5.4,亦无明显变化。因此,突变体菌株ΔVdku80的生长速率、产孢量和致病性相对于野生型菌株均未发生明显变化。使用野生型菌株作为基因敲除的受体菌株,几丁质合成酶编码基因ChsV和ChsVI的基因敲除转化子在总转化子中的比例分别为44%和31%;而使用ΔVdku80作为基因敲除的受体菌株,几丁质合成酶编码基因ChsV和ChsVI基因敲除转化子在总转化子中的比例分别为94%和87%。【结论】Vdku80缺失的菌株ΔVdku80作为受体菌株可以提高其他基因的基因敲除效率,且Vdku80的缺失对大丽轮枝菌的生长速率、产孢量和致病性均无影响,因此不会干扰其他基因的敲除所带来的表型变化。Vdku80缺失的菌株ΔVdku80可作为高效的大丽轮枝菌基因敲除受体菌株。