Immunohistochemical expression of p21 in normal tissues of salivary gland, pleomorphic adenoma and carcinoma ex pleomorphic adenoma-(undifferentiated and adenocarcinoma types)

Objective: Our study aimed to characterize alteration in the immunohistochemical expression of p21 in normaltissue of the salivary gland surrounding pleomorphic adenoma, the tumor cells of pleomorphic adenomas, andcarcinoma arising in pleomorphic adenoma.Study design: A selected series of 29 cases of pleomorphic adenomas, and 27 cases of carcinoma ex-pleomorphicadenoma (undifferentiated and adenocarcinoma types) were examined.Results: The results showed that p21 expression was negative in the most components of normal tissue of the salivary gland surrounding pleomorphic adenoma, 24 cases out of 29 of the non tumour duct cells (82.8%), and 28 (96.6%) cases out of 29 of the acinar cells shows negative p21 expression. P21 expression in pleomorphic adenomas shows that 2 cases out of 29 (6.9%) strongly expressed in the duct cells. p21 was strongly expressed incarcinoma cells in 9 (33.3%) cases out of 27.Conclusion: Our data suggest that the strong nuclear staining as an indicator for altered p21, then the alteration of p21 expression would increase from pleomorphic adenoma to carcinoma arising in pleomorphic adenomas (6.9%versus 33.3%). (AU)

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[Soluble expression, purification and bioactivity of recombinant human Era protein].

AIM: To prepare a soluble human Era(hEra) protein and to measure its bioactivity. METHODS: Human era cDNA gene from pUC19 plasmid was subcloned into the expression plasmid pMAL-p2x. pMAL-hEra was transducted to E.coli TB1 and the strain was induced by isopropyl beta-D-thiogalactopyranoside (IPTG). RESULTS: The expressed MBP-fused protein existed in a soluble form. The fused protein made up 23.9% of the total cell lysate. It was purified by amylose affinity chromotography and digested with Factor X. Although the fused segment was dissected, the remained hEra protein was unstable in the solution with the passage of time. The activity assay showed that hEra was a GTPase that could bind GTP and hydrolyze GTP to GDP. CONCLUSION: Human Era protein can be expressed in a soluble form and it has been proved to be a kind of G protein by the experiments in vitro. The study is important to further research into the function of human era gene.

Functional interactions between isolated SH2 domains and insulin/Ras signaling pathways of Xenopus oocytes: opposite effects of the carboxy- and amino-terminal SH2 domains of p85 PI 3-kinase.

Purified amino-terminal Src homology 2 (SH2) domains of GAP, PLCgamma1 and the p85alpha subunit of PI 3-kinase, as well as the carboxy-terminal SH2 domain of the latter protein and the unique SH2 domain of Grb2, were injected into full grown, stage VI Xenopus laevis oocytes. None of the injected domains showed any effect when injected alone, nor did they affect the rate of GVBD induced by progesterone, an adenylate cyclase-dependent process. On the other hand, the unique Grb2 SH2 domain and all N-terminal SH2 domains injected inhibited to various degrees the rate of insulin-induced GVBD, a tyrosine kinase dependent pathway. Interestingly, and in contrast to the behavior shown by the N-terminal domain of the same molecule, the C-terminal SH2 domain of p85 did not inhibit, but slightly accelerated the rate of GVBD induced by insulin. Furthermore, whereas the Grb SH2 domain and all N-terminal SH2 domains tested failed to co-operate with normal Ras protein to induce GVBD, the C-terminal SH2 domain of p85alpha exhibited significant synergy when coinjected with normal Ras protein, indicating that the C- and N-terminal SH2 domains of p85alpha exert opposite (positive and negative, respectively) regulatory roles in the control of oocyte insulin/Ras signaling pathways. Our results demonstrate that the purified, isolated SH2 domains retain structural and functional specificity and that Xenopus oocytes constitute an useful biological system to analyse their functional role in tyrosine kinase signaling pathways.

Histopathological studies of superficial-type early colorectal carcinoma.

BACKGROUND: Superficial-type early colorectal carcinoma (SCa) is presently not a rare finding and is very important in discussions regarding the development of large bowel cancers, although the histologic characteristics of SCa remain obscure. METHODS: Using 54 SCa lesions (34 intramucosal adenocarcinomas (SCa-m) and 20 adenocarcinomas with invasion to the submucosa (SCa-sm)), the largest dimension of the depressed region of the lesion and the greatest dimension of the entire lesion were measured by the computed image analyzer, and the expression of p53 and ras of SCa were examined immunohistochemically. RESULTS: The percent of depressed regions in SCa lesions measuring less than 5 mm in greatest extent was larger than in those measuring more than 6 mm, and the percent of depressed regions in SCa-sm with deeper carcinoma invasion was lower than that of SCa-sm with shallower invasion. There was a positive correlation between the depth of invasion and the maximum dimension of the carcinoma. The frequency of association with adenoma in all SCa-m was 21% and was 32% in SCa-m that were more than 6 mm in greatest extent, although all minute SCa-m lesions less than 5 mm were pure carcinomas without any adenomatous component. Positive expression of ras was noted in 41% of SCa-m and 36% of SCa-sm, respectively, while positive expression of p53 was noted in 63% of SCa-m and 88% of SCa-sm, respectively. CONCLUSIONS: These results suggested that 70% to 80% of SCa developed via a de novo carcinoma theory and showed the depression form in the initial histologic stage and thereafter in the flat-protrusion form, while 20% to 30% of SCa arose from the preexisting flat adenoma via the adenoma-carcinoma sequence theory. The results also suggested that p53 was related to the enlargement and deeper invasion of SCa, regardless of the sequence of development of colorectal cancer.

Neoplastic transformation of a human prostate epithelial cell line by the v-Ki-ras oncogene.

Investigations of mechanisms of human prostate carcinogenesis are limited by the unavailability of a suitable in vitro model system. We have demonstrated that an immortal, but nontumorigenic, human epithelial cell line (267B1) established from fetal prostate tissue can be malignantly transformed by a biological carcinogen, and can serve as a useful model for investigations of the progression steps of carcinogenesis. Activated Ki-ras was introduced into 267B1 cells by infection with the Kirsten murine sarcoma virus. Morphological alterations and anchorage-independent growth were observed; when cells were injected into nude mice, poorly differentiated adenocarcinomas developed. These findings represent the first evidence of malignant transformation of human prostate epithelial cells in culture, and support a role for Ki-ras activation in a multistep process for prostate neoplastic transformation.

Modulation of guanine triphosphate nucleotide binding to p21ras immunoprecipitates of rat liver plasma membranes by agents affecting redox state.

GTP-gamma-[35S] and GTP-gamma-[32P] or GTP-alpha-[32P] bound to plasma membranes of rat liver was immunoprecipitated using anti p21V-H-ras. Binding was enhanced approximately 2-fold by incubation with an exogenous electron acceptor, potassium ferricyanide (but not with potassium ferrocyanide), or oxidized ubiquinone10 and was inhibited or unaffected by incubation with reduced pyridine nucleotides (NADH or NADPH) or reduced ubiquinone10. The results suggest a mechanism of guanine nucleotide exchange that is responsive to oxidation-reduction.

Free L-phosphotyrosine activates human platelets: molecular evidence for a new signal transducer.

Human platelets, either purified or plasma-enriched, are activated when exposed to free L-phosphotyrosine. Physical aggregation is similar to that induced by collagen, although with distinctive biochemical features. Among these, a mobility shift of a GTP-binding protein specifically recognized by an anti-Ki-v-ras monoclonal antibody and an altered pattern of low molecular weight phosphorylated polypeptides are the most outstanding features. Since free phosphotyrosine is detected in platelets extracts, its role as a new signal transducer as well as its putative modulating action over protein phosphorylation are discussed.

Asparagine 26, glutamic acid 31, valine 45, and tyrosine 64 of Ras proteins are required for their oncogenicity.

Ras and Rap1 proteins are related GTP-dependent signal transducers which require Gly-12, the effector domain (residues 32-40), and Ala-59 for stimulation of their GTPase activities by GAP1 and GAP3, respectively. The replacement of Gly-12 by Val or Ala-59 by Thr potentiates the Ras oncogenicity and Rap1A antioncogenicity. However, the mutations in the effector domain, in particular the replacement of Thr-35 by Ala, abolish both Ras oncogenicity and Rap1A antioncogenicity, indicating that the effector domain is involved in interactions of these signal transducers with their targets as well as the GAPs. In this paper, we demonstrate that (i) replacement of Tyr-64 of the Ha-Ras protein or Phe-64 of the Rap1A protein by Glu or other non-hydrophobic amino acids reduces their intrinsic GTPase activities and abolishes their stimulation by GAP1 or GAP3, respectively, (ii) replacement of Tyr-64 by Gly and other non-hydrophobic amino acids results in complete loss of the oncogenicity of the v-Ha-Ras protein, indicating that the hydrophobic residue 64, in addition to the known effector domain, is essential for the Ras protein to interact with its target as well as GAP1. In addition we have found that Asn-26, Glu-31, and Val-45 of the v-Ha-Ras protein are required for its oncogenicity. Replacement of the Ras residues at either positions 26, 31, or 45 by the corresponding Rap1A residues abolishes the Ras oncogenicity.

Production and characterization of anti-RAS p21 monoclonal antibodies.

Monoclonal antibodies (MAb) Ras 10 and Ras 11 were raised to an activated human Harvey-ras p21 and shown to react with recombinant p21 as well as p21 derived from human and rodent cells. Characterization studies by ELISA, immunoprecipitation and Western blot procedures demonstrated that MAb Ras 10 (IgG2a) and Ras 11 (IgG2b) react with normal p21, activated p21, and p21 from each of the Harvey, Kirsten and N-ras families. Studies illustrated that MAb Ras 10 and Ras 11 can also be used in flow cytometry and immunohistochemistry to specifically detect cellular p21. ELISA, immunoprecipitation and Western blot studies comparing rat anti-p21 MAb Y13-259 with Ras 10 and Ras 11 demonstrated that Ras 10 and Ras 11 had a greater sensitivity for ras protein detection than Y13-259. Collectively, these studies illustrate that MAb Ras 10 and Ras 11 can be applied to a variety of assay formats to detect ras proteins and, therefore, may be valuable tools in detecting and measuring of ras protein expression in normal, neoplastic and pre-neoplastic cells.

EJ/ras neoplastic transformation of simian virus 40-immortalized human uroepithelial cells: a rare event.

To determine if expression of mutant p21 ras could convert Simian Virus 40-immortalized human uroepithelial cell line (SV-HUC) to tumorigenicity, SV-HUC cells were transfected with pSV2-neo (a neomycin-resistant gene) or PREJ/ras (c-HA-ras-1 with the 12th codon mutation and neo). Seven independent G418-resistant clones (A----G) were isolated from each group (SV-HUC/ras and SV-HUC/neo). SV-HUC/ras clones were morphologically altered, while SV-HUC/neo clones retained a typical SV-HUC epithelial morphology. Electrophoretic analysis of immunoprecipitated ras proteins detected altered p21 ras protein in four of seven SV-HUC/ras clones at passage (P)2 and in five of seven clones at P12 posttransfection. The relative levels of ras p21 differed among the clones and appeared to increase with passage in culture. RNA and DNA dot blot analyses showed that clones with more abundant mutant p21 also had higher ras RNA levels and, in one case, increased ras gene copy number. No altered ras protein was detected in any SV-HUC/neo clones. ras- and neo-transfected clones were tested for tumorigenicity at P2 posttransfection and again at P12 by four s.c. inoculations each into athymic nude mice. None of 56 inoculations of SV-HUC/neo clones was tumorigenic. None of the SV-HUC/ras clones at P2 gave rise to tumors at all four injection sites. However, two ras-transfected clones, SV-HUC/ras-B and SV-HUC/ras-F, produced one tumor each. One clone, SV-HUC/ras-D which produced abundant mutant p21, was negative when inoculated at P2, but produced tumors in four of four sites when reinoculated after ten passages in vitro. All tumorigenic clones had detectable levels of mutant ras p21. However, the relative levels of altered p21 ras protein among the SV-HUC/ras clones did not directly predict their tumorigenic potential, as several nontumorigenic SV-HUC/ras clones had protein levels equal to or higher than the most tumorigenic clone (SV-HUC/ras-D at P12). Cell lines established from the tumor explants exhibited higher ras gene copy numbers, higher RNA levels, and more abundant p21 than was seen in the clones at the time of inoculation. Therefore, increases in ras protein abundance occurred during tumor formation in vivo, as well as during passage of cells in culture, and such cells apparently had a selective growth advantage. However, expression of abundant mutant ras protein was not in itself sufficient for neoplastic transformation of SV-HUC.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of the ras p21 GTPase-activating protein-stimulated GTPase activity of c-Ha-ras p21 by smg p21 having the same putative effector domain as ras p21s.

ras p21 GTPase-activating protein (GAP) has been proposed to interact with the putative effector domain of ras p21s, and smg p21, a ras p21-like guanine nucleotide binding protein (G protein), has been shown to have the same amino acid sequence as ras p21s in this region. In the present studies, we examined the effects of ras p21 GAP on the GTPase activity of smg p21 purified from human platelets, of smg p21 on the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21 purified from Escherichia coli, and of c-Ha-ras p21 on the smg p21 GAP1- or -2-stimulated GTPase activity of smg p21. ras p21 GAP stimulated the GTPase activity of c-Ha-ras p21 but not that of smg p21. The GTP-bound form of smg p21, however, inhibited the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21 in a dose-dependent manner. The half-maximum inhibition by smg p21 was obtained at 0.4 microM which was more potent than previously observed for ras p21 (2-200 microM). The GDP-bound form also inhibited the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21, but the efficiency was 40-50% that of the GTP-bound form. smg p21 GAP1 and -2 stimulated the GTPase activity of smg p21 but not that of c-Ha-ras p21. c-Ha-ras p21 did not inhibit the smg p21 GAP1- or -2-stimulated GTPase activity of smg p21. These results indicate that ras p21 GAP interacts with smg p21 without the subsequent stimulation of its GTPase activity.

p21ras is modified by a farnesyl isoprenoid.

Association of oncogenic ras proteins with cellular membranes appears to be a crucial step in transformation, ras is synthesized as a cytosolic precursor, which is processed to a mature form that localizes to the plasma membrane. This processing involves, in part, a conserved sequence, Cys-Ali-Ali-Xaa (in which Ali is an amino acid with an aliphatic side chain and Xaa is any amino acid), at the COOH terminus of ras proteins. Yeast a-factor mating hormone precursor also possesses a COOH-terminal Cys-Ali-Ali-Xaa sequence. However, while the COOH-terminal cysteine has been implicated as a site of palmitoylation of ras proteins, in mature a-type mating factor this residue is modified by an isoprenoid, a farnesyl moiety. We asked whether the Cys-Ali-Ali-Xaa sequence signaled different modifications for the yeast peptides (farnesylation) than for ras proteins (palmitoylation) or whether ras proteins were similar to the mating factors and contained a previously undiscovered isoprenoid. We report here that the processing of ras proteins involves addition of a farnesyl moiety, apparently at the COOH-terminal cysteine analogous to the cysteine modified in the yeast peptides, and that farnesylation may be important for membrane association and transforming activity of ras proteins.

Expression and characterization of the Ha-ras p21 protein produced at high levels in the insect/baculovirus system.

Normal and mutated cDNAs of Ha-ras have each been cloned into a standard (pAc373) and a novel (p36C) baculovirus transfer vector and introduced via homologous recombination into the genome of Autographa californica nuclear polyhedrosis virus immediately downstream of the polyhedrin promoter. Spodoptera frugiperda cells infected with recombinant virus containing the normal Ha-ras gene express very high levels of ras p21 protein (approximately 20% of total cell protein), whereas the mutant protein was expressed at considerably lower levels. Molecular analysis showed that this was most likely due to a post-transcriptional event. The expression vector p36C produced considerably higher levels of recombinant p21 compared to the more commonly used pAc373. The majority of the normal ras p21 protein is soluble, cytoplasmic, and appears to be nonacylated. However, about 10% of the p21 associates with the membrane fraction of infected cells and migrates as a slightly faster band on gels. Furthermore, this band is sensitive to hydroxylamine treatment and shows specific incorporation of [3H]palmitate, strongly suggesting that it is the palmitoylated form of p21, which is the biologically active form of the protein. Both the soluble and membrane-associated p21 have been purified to homogeneity under nondenaturing conditions, the latter in the presence of detergents. The isolation of native palmitoylated p21 has not been reported previously. The difference in hydrophobicity between these two proteins has been demonstrated by Triton X-114 partitioning. The use of the insect/baculovirus expression system to express relatively high levels (20 mg/liter) of palmitoylated p21 should aid experiments to resolve the structural and functional properties of this molecule.

Cotransfection of plasmids with ras and myc oncogenes to diploid cells derived from rodent fetuses: alteration of neoplastic transformation frequency depending on the gestation period.

To analyze the mechanism of neoplastic transformation of rodent diploid cells by ras and myc oncogenes, human EJ c-Ha-ras and mouse c-myc second and third exons promoted by SV40 promoter were connected to pSV2neo and pSV2gpt, respectively. Mouse and rat primary fetal cells cotransfected with both genes formed transformed and nontransformed colonies in a medium containing G418 and mycophenolic acid (MPA). The proportion of transformed colonies in the total G418/MPA-resistant colonies decreased dependent on the stage of the gestation period of rat fetuses from which primary cells had been obtained. Analysis of randomly isolated colonies showed that the transformed colonies had a high copy number and high amount of expression of the introduced genes, were anchorage independent, and were tumorigenic in nude mice. On the other hand, the nontransformed colonies had a low copy number, low amount of expression, and no tumorigenicity. This contrast indicated not only that the activated Ha-ras and myc oncogenes had been integrated, but also that the amplification or overexpression (or both) of these genes was required for the rodent diploid cells to be transformed. We conclude that early-stage rat fetal cells might have endogenous factors that promote cell transformation. Alternatively, late-stage cells might have factors that suppress cell transformation by activated Ha-ras and myc oncogenes.

Expression of ras p21 oncogene product on human bladder tumors.

The immunohistochemical localization of ras p21 oncogene product was examined in human bladder cancer. These bladder cancers consist of 52 superficial bladder tumors and 10 cases of invasive tumor. Five (9.6%) of 52 superficial tumors were demonstrated to be positive for ras p21 product. Two (20%) of 10 cases which showed deep tumor infiltration were demonstrated to be positive for ras p21 oncogene product. When we analyzed the incidence of positive staining for ras p21 with regard to histological grade of bladder cancer, 2 of 30 (7%) patients with G1, 4 of 26 (15%) with G2, and 1 of 6 (17%) with G3 had positive staining for the ras p21 oncogene product. One of five patients who showed positive staining on tumors had tumor recurrence 3 months later. The remaining 4 patients positive for the ras p21 oncogene product had no tendency for recurrence in 11-19 months' follow-up. Therefore, the incidence of detection of ras p21 oncogene product on superficial and deep infiltrating bladder tumor was similar and there was no significant correlation between the incidence of positive ras p21 staining and depth of invasion of bladder tumors or histological grade.