Microinjection of a p21ras antibody into PC12 cells inhibits neurite outgrowth induced by nerve growth factor and basic fibroblast growth factor

The role of p21ras in signal transduction in PC12 cells was studied using an antibody that blocks its function. Native cells were microinjected with either a control solution or a solution containing the monoclonal antibody Y13-259. Treatment of the cells with growth factors appeared to enhance the ability of the cells to survive the microinjection procedure. Of the cells microinjected with the control solution 66-69% of those treated with either nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) were still present 24 h post-injection, compared with only 57% for those not treated with growth factor after microinjection. This effect of the growth factors was inhibited by introduction of the Y13-259 antibody, suggesting that it occurs through a pathway that involves p21ras. Similarly, introduction of the Y13-259 antibody into cells also resulted in a statistically significant decrease in the percentage of neurite-bearing cells; 25-36% of the cells microinjected with the control solution had neurites, whereas 12-14% of the cells microinjected with the antibody solution had neurites. This decrease suggests that the induction of neurite outgrowth and the maintenance of established neurites by these growth factors is dependent on a functional p21ras pathway. As well as complementing the finding that p21ras is apparently involved in the mechanism of action of NGF in PC12 cells, these results further establish (1) that p21ras is also involved in the mechanism of action of bFGF, and (2) that the effect of NGF and bFGF on the number of labeled cells still present 24 h postinjection requires a functional p21ras protein.     

Microinjection of a p21ras Antibody into PC12 Cells Inhibits Neurite Outgrowth Induced by Nerve Growth Factor and Basic Fibroblast Growth Factor

Abstract

The role of p21ras in signal transduction in PC12 cells was studied using an antibody that blocks its function. Native cells were microinjected with either a control solution or a solution containing the monoclonal antibody Y13-259. Treatment of the cells with growth factors appeared to enhance the ability of the cells to survive the microinjection procedure. Of the cells microinjected with the control solution 66-69% of those treated with either nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) were still present 24 h post-injection, compared with only 57% for those not treated with growth factor after microinjection. This effect of the growth factors was inhibited by introduction of the Y13-259 antibody, suggesting that it occurs through a pathway that involves p21ras. Similarly, introduction of the Y13-259 antibody into cells also resulted in a statistically significant decrease in the percentage of neurite-bearing cells; 25-36% of the cells microinjected with the control solution had neurites, whereas 12-14% of the cells microinjected with the antibody solution had neurites. This decrease suggests that the induction of neurite outgrowth and the maintenance of established neurites by these growth factors is dependent on a functional p21ras pathway. As well as complementing the finding that p21ras is apparently involved in the mechanism of action of NGF in PC12 cells, these results further establish (1) that p21ras is also involved in the mechanism of action of bFGF, and (2) that the effect of NGF and bFGF on the number of labeled cells still present 24 h postinjection requires a functional p21 ras protein.     

Interleukin (IL)-2 activation of p21ras in murine myeloid cells transfected with human IL-2 receptor beta chain.

The T cell growth factor interleukin-2 (IL-2) induces p21ras activation in T lymphocytes. To determine whether the IL-2 receptor (IL-2R) can regulate p21ras when expressed in a non-T cell environment we have examined the ability of IL-2 to activate p21ras in 32D murine myeloid progenitor cells transduced with human IL-2R beta chains. These cells are denoted beta 53 cells. 32D cells normally proliferate in response to IL-3 but the expression of the IL-2R beta chain confers IL-2 responsiveness to the cells. Our data show that IL-3 is able to activate p21ras in the parental 32D cells and both IL-2 and IL-3 can stimulate p21ras in the IL-2R-expressing beta 53 clone of 32D. In T lymphocytes, activation of protein kinase C (PKC) with phorbol esters is sufficient to stimulate p21ras. However, in 32D and beta 53 cells activation of PKC with phorbol esters does not result in p21ras activation even though these cells express functional PKC. It appears, therefore, that a PKC-mediated pathway for p21ras regulation exists in T lymphocytes but not in 32D cells. The IL-2R can couple to p21ras independently of the concomitant presence of the PKC pathway for p21ras regulation. These data imply that multiple intracellular mechanisms may exist to regulate p21ras and that cells of different lineages may differ with regard to p21ras regulation.     

The role of p21ras in pancreatic neoplasia and chronic pancreatitis.

K-ras mutations have been detected in both ductal cell carcinoma and intraductal papillary mucinous tumor (IPMT) of pancreas. The frequency of this mutation in ductal cell carcinoma is high, whereas in IPMT, it is variable. It has been suggested that the relatively high frequency of this mutation in ductal cell carcinomas compared with IPMT may account for the differences in biological behavior between these tumor types. More recently, the significance of K-ras mutations in pancreatic tissue has been questioned with the demonstration of this mutation in nonneoplastic pancreata. The current study aims to estimate the relative frequency and evaluate the biological significance of K-ras gene mutations in these neoplasms by performing polymerase chain reaction (PCR) assays of microdissected areas of IPMT, ductal cell carcinomas, and resected chronic pancreatitis. The study also investigates whether alterations of p21ras occur in K-ras mutation-negative cases by using immunohistochemical staining for K-, N- and H-ras. K-ras codon 12 mutations were found almost as frequently in IPMT (71%) as in ductal cell carcinomas (78%). They were also associated with the earliest morphological lesion, flat mucinous change. This mutation also was detected in 42% of cases of chronic pancreatitis. Expression of p21ras was found to correlate closely with K-ras mutation status in IPMT and ductal cell carcinoma. Negative staining for pan-ras, H-ras, and N-ras in cases with wild-type K-ras genes suggests that alternative routes of ras gene alteration are not operative in IPMT or ductal carcinoma. The findings suggest that K-ras activation is frequently associated with both IPMT and ductal cell carcinoma. Its high prevalence in nonneoplastic pancreata suggests that it is also associated with self-limited morphological lesions of the pancreas that do not progress to malignancy.     

Expression of ras proto-oncogene related protein p21 in normal human skin and cutaneous tumours.

The cellular proto-oncogene, ras, is known to play an important role in the regulation of cell growth and proliferation in normal and malignant conditions. The present study was undertaken to immunohistochemically examine the expression of ras protooncogene product p21 in normal human skin and some cutaneous tumours. In normal skin, the expression of p21 was found in sweat glands, sebaceous glands, capillary endothelium, and smooth muscles, while epidermis was devoid of reaction product. Keratoacanthoma and the granular cells of verruca vulgaris were immunoreactive to the antibody for p21. Bowen's disease and squamous cell carcinoma were positive for p21, but basal cell carcinoma and seborrheic keratosis were negative. In mammary and extramammary Paget's diseases, the immunoreactivity was inconsistent. The expression of p21 in malignant melanoma cells was intense, whereas normal melanocytes and nevus cells were devoid of the expression. These results suggest that the expression of p21 does not correlate with nuclear anaplasia and malignant behaviour of cutaneous tumours.     

The regulation and function of p21ras in T cells.

In T cells, activation of the guanine-nucleotide-binding proteins encoded by the p21ras proto-oncogenes is a common response to triggering of the T-cell antigen receptor, the adhesion molecule CD2 and the receptor for the cytokine interleukin 2. This article by Julian Downward and colleagues describes the mechanisms of p21ras regulation and the potential function of p21ras in T cells, and discusses the evidence that multiple intracellular pathways may be involved in the coupling of cell surface receptors to p21ras.     

Expression of EGF, EGF-receptor, p53, v-erb B and ras p21 in colorectal neoplasms by immunostaining paraffin-embedded tissues

Immunohistochemical studies were performed to clarify the significance of the expression or overexpression of epidermal growth factor (EGF), EGF-receptor (EGFR), p53, v- erb B, ras p21 in 23 cases each of tubular adenoma and adenocarcinoma. The expression of EGF, EGFR, p53, v- erb B, and ras p21 in paraffin-embedded tissues, from 46 patients with colorectal tumors (adenoma: 23 cases; 14 mild dysplasia, six moderate dysplasia, three severe dysplasia, adenocarcinoma: 23 cases; 17 well differentiated, two moderately differentiated, three poorly differentiated, one mucinous carcinoma was analyzed immunohistochemically using anti-EGF, EGFR, p53, v- erb B and ras p21 antibodies. The EGF and ras p21 tended to express more strongly in carcinoma cases than in the adenoma cases, and in severe and moderate dysplasia than in mild dysplasia (EGF: stained positive in five adenomas [21.74%] and 17 adenocarcinomas [73.91%]; ras p21: stained positive in six adenomas [26.09%] and 14 adenocarcinomas [60.87%]. The EGFR stained positive in two adenomas (8.70%) and two adenocarcinomas (8.70%). The p53 and v- erb B showed positive staining only in the carcinoma cases (p53: stained positive in four cases [17.39%]; v- erb B: stained positive in eight cases [34.78%]). This study suggests that these factors seem to have some role in the progression of colon neoplasms. It suggests that genetic alteration is not always equal to the overexpression of protein products, but that it reflects them well, and that the staining makes some contribution to differential diagnosis in colorectal neoplasms.     

Expression of ras oncogene p21 in prostate cancer

The major neoplastic transformation-inducing genes of human solid tumors are members of the ras oncogene family. We used an immunohistochemical assay to assess expression of both the unaltered and the mutated ras oncogene protein (p21) in normal and neoplastic prostatic cells. With the concentration of monoclonal antibody used in this study, epithelial and stromal cells from subjects with normal prostates and from 19 patients with benign prostatic hyperplasia were negative for p21 antigen. This antigen was detected in 2 of 6 prostates with Grade I carcinoma, 4 of 6 with Grade II, and all of 17 with higher grades. A semiquantitative immunohistochemical method demonstrated that expression of the p21 antigen in a carcinoma strongly correlated with nuclear anaplasia and was inversely related to the degree of glandular differentiation. However, markedly anaplastic tumors were often more heterogeneous in expression of p21 and contained areas of low staining for the antigen. Comparison of p21 antigen with tumor carcinoembryonic antigen and prostate-specific antigen demonstrated that ras p21 was the only phenotypic marker that correlated with histologic tumor grade. Thus, ras oncogene p21 may represent a new class of biologically relevant tumor markers and may be a useful adjunct to histopathologic examination in determining the prognosis of patients with prostate cancer.     

Immunocytochemical demonstration and significance of p21 ras family oncogene product in benign and malignant breast disease

It has been suggested that the immunocytochemical demonstration of the p21 ras oncogene product is a useful marker of malignancy in breast disease. We have studied the reactivity of a series of specimens of benign and malignant breast disease with the anti ras p21 monoclonal antibody Y13-259, and shown widespread positive staining in both benign and malignant (including metastatic) disease as well as in adjacent 'normal' epithelium. In addition some staining of stromal cells as well as nerve fibres was observed. Our results suggest that the presence of ras p21 protein as demonstrated by this antibody is not a useful marker of malignancy or of proliferating epithelium but is rather a normal feature of certain cell types.     

Optimal preservation of p21 ras immunoreactivity and morphology in paraffin-embedded tissue

Specific immunostaining of p21 ras protein by the well-characterized pan-ras antibody Y13-259 is achieved in paraffin sections of human and animal tissues fixed in periodate-lysine-paraformaldehyde-dichromate (PLPD). Intensity of staining is as good as in cryostat sections, with superior histological detail. Localization to plasma membrane is demonstrated in rodent cells genetically manipulated to express abundant p21 ras (the FHO5T1 cell line), both in preparations suspended in agar after culture in vitro and in those growing as tumour in vivo. Strong positive staining is observed in neoplasms of human breast and colon, tissues in which there is independent evidence of elevated ras gene expression. The superior morphology afforded by this technique allows clear characterization of p21 ras expression in small premalignant lesions for which other methods of detection of oncogene expression are not appropriate.     

Decreased p21 levels in anti-sense ras transfectants augments NK sensitivity

We have shown that the oncogenic EJ-ras gene, under the control of a metallothionein-I (Mt) promoter, can be induced to cause an increased susceptibility of transfected 10T1/2 fibroblasts to cytolysis mediated by natural killer (NK) cells. This effect may be specific to the ras gene family, since other oncogenes that we have tested here (src) and elsewhere (myc) do not show this effect. We have now examined the effect of modulating the level of p21 in both a positive or negative manner. The level of p21 ras was decreased by two independent mechanisms. First Zn2+ was removed from Mt-EJ-ras transformed cells. In the second approach we transfected 10T1/2 cells with a Mt-anti-sense c-H-ras construct which reduced p21 expression, slowed the growth rate and altered the morphology of 10T1/2 cells when induced with Zn. Surprisingly, the decrease in p21 ras levels by both approaches caused a marked increase in NK susceptibility (NKS) which was equivalent to that observed when the p21 ras levels were increased either by inducing EJ-ras or removing Zn2+ from Mt-anti-sense c-H-ras containing cells. The kinetics of induction of NK sensitivity due to decreasing normal p21 ras levels was identical to that observed for increasing mutated p21 ras levels. Peak enhancement of NKS was observed 24 hr after ras perturbation. These results suggest that either a positive or negative change in the steady-state level of p21 ras is sufficient to induce NK sensitivity, and NK sensitivity is not inextricably linked to cellular transformation by the ras gene.     

Involvement of p21ras activation in T cell CD69 expression

The involvement of p21^ras in the induction of the early activation antigen CD69 was investigated in T cells. Expression of a v-Ha-ras coding for a constitutively active ras protein in Jurkat cells resulted in CD69 induction on the cell surface. Transfected ras was shown to be constitutively activated and functionally efficient, since it could be immunoprecipitated in the guanosine triphosphate (GTP)-bound form and it induced transactivation of an AP-1 consensus-chloramphenicol acetyltransferase reporter gene. The requirement for ras activation in T cell receptor (TcR) CD3-mediated CD69 induction was also investigated. The expression of a dominant negative c-Ha-ras-N17 mutant markedly reduced the amount of GTP that could be immunoprecipitated from ras proteins after TcR/CD3 triggering in Jurkat cells, and concomitantly decreased TcR/CD3-mediated CD69 induction. These results suggest a central role for ras in TcR/CD3-mediated CD69 expression in T cells.     

Distribution of p21ras in postimplantation rat embryos.

Expression of ras cellular oncogenes during the early postimplantation period in the rat was investigated using immunohistochemistry to p21ras. A broad spectrum polyclonal antibody recognizing N-, Ha- and Ki- forms of p21ras was used in an indirect avidin-biotin-peroxidase (ABC) technique. Positive staining indicating the presence of p21ras was found in embryos from 6.5 to 12 days embryonic age. In early egg cylinders (6.5 days), positive staining for p21ras was observed on the ectoplacental cone, primitive ectoderm and trophectoderm, while primitive endoderm and parietal endoderm appeared paler. In later egg cylinder stages (7.5 days), strong positive staining was observed in the primitive embryonic ectoderm and ectoplacental cone, but parietal and visceral endoderm still appeared to be devoid of positive staining. As development proceeded during primitive streak stages, the visceral and parietal endoderm became positively stained. By 10 days, all tissues appeared to be positive for p21ras, with strong staining appearing in the heart and neural elements. Therefore, p21ras does not appear to be ubiquitous in the rat conceptus prior to gastrulation, but shows differential distribution, appearing later in endodermal derivatives. Possibly p21ras is involved in determination of the ectodermal and endodermal lineages.     

ras p21 oncoprotein expression in human colonic neoplasia—an immunohistochemical study with monoclonal antibody RAP-5

Expression of the ras oncogene product p21 (ras p21) in benign and malignant human colonic tissues was studied using the monoclonal antibody RAP-5 and the avidin-biotin-peroxidase technique. Histologically normal colonic mucosa and hyperplastic mucosa adjacent to carcinomas (transitional mucosa) were found, in most cases, to be negative for reactivity with the antibody or showed weak staining of a few epithelial cells. Similar findings were observed in hyperplastic and juvenile polyps. Of the 145 adenomas studied, 47 (32.4%) showed detectable levels of ras p21 expression. RAP-5 immunohistochemical staining was significantly associated with the degree of epithelial dysplasia (P less than 0.01) and the size of adenoma (P less than 0.05), but not with the histological type. Fifty-four of 70 primary adenocarcinomas (77.1%) were reactive with RAP-5 and usually demonstrated a higher percentage of stained cells and more intense cytoplasmic staining than that observed in adenomas. Although metastases often displayed a similar or even higher levels of ras p21 expression compared with the primary carcinomas, in 10 cases one or more metastatic lesions showed lower levels of ras p21. These results suggest that enhanced ras p21 expression may, at times, occur in the early stages of human colon carcinogenesis but are probably not associated with metastatic tumour progression.     

Defective activation of p21ras in peripheral blood mononuclear cells from patients with insulin dependent diabetes mellitus.

We previously reported that a decreased TCR mediated activity of the GTP-GDP binding p21ras protooncogene is associated with prediabetes in non-obese diabetic (NOD) mice. Furthermore, prevention of autoimmune diabetes is associated with reversal of the p21ras signaling defect in NOD T cells. Based on these animal studies we determined the activation of p21ras in PBMC from patients with Insulin Dependent Diabetes Mellitus (IDDM), Non-Insulin Dependent Diabetes Mellitus (NIDDM) and normal healthy controls. Stimulation by PHA induced a decrease of 3.7 +/- 1.4% and an increase of 2.44 +/- 2.3%, p < 0.02 and 2.6 +/- 1.6%,p < 0.003 in the basal unstimulated p21ras activity in the IDDM, NIDDM and normal control groups, respectively. Expression of p21ras and its regulatory elements, the GTPase activating protein p120ras-GAP and the guanine nucleotide releasing factor (GNRF) hSOS, was comparable in the three groups. The in vitro proliferative response to PHA was comparable in the IDDM and control groups: stimulation index (SI) of 8.6 +/- 2.5 and 9.4 +/- 3.5 respectively, p < 0.44. No correlations were found in the IDDM patients between the degree of p21ras activation and the mitogen induced in vitro proliferative response or the various clinical parameters including age, gender, disease duration, daily insulin requirements and metabolic control. Taken together these data indicate that PBMC from IDDM patients are characterized by a persistent impairment in the activation of their p21ras. They also suggest that p21ras stimulated activity is a sensitive and independent parameter of PBMC activation in these patients.     

Immunoprecipitation of cell lysates with RAP-5 does not specifically detect ras oncogene product p21

In the process of developing accurate quantitation of the ras protein (p21), we have screened available anti-ras antibodies for their utility in immunoprecipitation. Immunoprecipitation with the anti-ras antibody RAP-5 consistently failed to precipitate p21 present in two different cell lines (HSIC-5 and MCF-7), but did precipitate numerous other proteins present in these cell lines. Specificity in immunoprecipitation could not be achieved by varying the concentration of RAP-5. In addition, immunohistochemical staining of the nuclei of occasional polymorphonuclear leukocytes is seen, further supporting the contention that RAP-5 is binding to proteins other than ras p21. We conclude that while RAP-5 may recognize an epitope present on the ras protein, this epitope also appears to be present on a wide variety of other cellular proteins and, as such, RAP-5 is of no use in the immunoprecipitation of p21.     

Microinjected ras family oncogenes stimulate DNA synthesis in quiescent mammalian cells

Oncogenes of the ras family stimulate DNA synthesis when microinjected into quiescent mouse and hamster fibroblasts, as detected by in situ autoradiography. The molecularly cloned genomes of Harvey and Kirsten sarcoma viruses, the cloned Harvey ras gene, and the product of the v-ras gene, the p21v-rasH protein, stimulate DNA synthesis in quiescent cells. This stimulation is comparable to the stimulatory activity of the microinjected SV40 T-antigen-coding gene. The demonstration that these oncogenes can stimulate transient DNA synthesis in quiescent cells is relevant to understanding the mechanism by which these genes are able to transform cells in vitro and induce tumors in animals.     

Memory T cells of a patient with follicular thyroid carcinoma recognize peptides derived from mutated p21 ras (Gln-->Leu61).

Point mutations in ras genes resulting in substitutions of amino acid Gly in positions 12 and 13, and Gln in position 61 of the ras gene product p21, are commonly found in human tumors. Peptides derived from aberrant p21 may elicit a tumor specific T cell response, provided that these peptides can bind to HLA molecules of the tumor and the patient has T cells able to recognize the corresponding peptide-HLA complex. Here we report that CD4+ T cells of memory type (CD45RO+) from a patient with a follicular thyroid carcinoma respond against a synthetic peptide derived from aberrant p21 ras having a Gln-->Leu substitution at position 61. Such responses were not observed when T cells from healthy volunteers or cancer patients where this mutation does not usually occur were stimulated with this peptide. The responding T cells did not cross-react with the corresponding peptide derived from native p21 ras nor did they recognize peptides carrying other substitutions in position 61. T cells clones were generated which recognized this Leu61 peptide when presented by HLA-DQ8 molecules. These T cell clones also recognized the corresponding intact p21 ras protein. By using several different synthetic peptides, a peptide with optimal stimulatory capacity was defined. Performing polymerase chain reaction and oligonucleotide probing we were, however, not able to detect the p21 ras gene encoding the Gln-->Leu substitution in DNA from tumor biopsies from the patient. This may indicate that tumor cells harboring the mutation leading to the Gln-->Leu substitution had been eliminated and that tumor progression was due to cells that had deleted the mutated ras gene. The finding that ras derived peptides and recombinant mutated p21 ras are immunogenic in man may form the basis for the development of cancer immunotherapy based on synthetic oncogene derived peptides.     

HPV16,18E6蛋白与p21ras,p53在食管癌组织中表达

采用ＳＰ免疫组化法对５２例食管鳞状细胞癌和３０例食管粘膜慢性炎（对照组）进行高危ＨＰＶ１６、１８Ｅ_６和ｐ２１ｒａｓ、ｐ５３癌基因产物的检测。结果表示：鳞癌组中Ｅ_６的阳性率为６７．３１％，与对照组相比差异有极显著性（Ｐ＜０．００１），其中Ｅ_６与ｐ５３呈双阳性者为５５．７７％（２９／５２）、８９．６６％（２６／２９），显示两者阳性着色出现在部分相同区域同一癌细胞核内（似表明Ｅ_６可与ｐ５３结合形成复合物从而导致野生型ｐ５３的降解）。本组ｐ２１ｒａｓ与ｐ５３、ｐ５３与Ｅ_６的阳性表达均具相关性（Ｐ＜０．０５）。提出ＨＰＶ１６、１８感染与本地区食管癌病因学密切相关，Ｅ_６抗体是诊断ＨＰＶ１６、１８感染的良好标记。