Immunocytochemical localization of ras p21 product in thyroid follicular cells of normal rats using monoclonal antibody RAP-5

Summary The ultrastructural localization ofras p21 product was studied immunocytochemically in thyroid follicular cells of normal rats using pre-embedded peroxidase-labelled antibody techniques and a monoclonal antibody, RAP-5, which had been raised against a synthetic peptide corresponding to amino acid positions 10–17 of theras p21 protein. Theras p21 product was detected in cisternae of rough endoplasmic reticulum, Golgi apparatus and the subapical portion of apical plasma membrane, in which it was most concentrated. This study indicated that the p21 product may be synthesized in the rough endoplasmic reticulum and finally localized at the subapical portion of the thyroid follicular cells, and also that the apical plasma membrane may be a major site for the reception of environmental stimuli.     

Oligomeric structure of p21 ras proteins as determined by radiation inactivation

Using radiation inactivation we determined that p21 ras proteins exhibit an oligomeric target size when assayed both structurally and functionally. Similar target sizes of p21 in ras-transformed cells and in purified preparations of the protein suggested that its structure is homo-oligomeric. p21 monomers were destroyed by radiation with the same target size as the GTP binding activity, indicating the occurrence of a tight association allowing energy transfer between the monomers. Irradiation in the presence of GTP, dithiothreitol, or EDTA did not change the target size. Normal (Gly12) and transforming (Lys12) forms of the protein exhibited similar target sizes. The homo-oligomeric structure suggests that p21 ras proteins do not conform to the structure of monomeric alpha subunits in classical G proteins (alpha beta gamma heterotrimers) and establishes similarities with other homo-oligomeric proteins (such as Escherichia coli CRP) which acquire the active conformation through subunit reorientation upon nucleotide binding.     

Monoclonal antibody Y13-259 recognizes an epitope of the p21 ras molecule not directly involved in the GTP-binding activity of the protein.

The p21 products of ras proto-oncogenes are GTP-binding proteins with associated GTPase activity. Recent studies have indicated that ras p21 may be required for the initiation of normal cell DNA synthesis, since microinjection of a monoclonal antibody, Y13-259, blocks serum stimulation of DNA synthesis in quiescent cell cultures (L. S. Mulcahy, M.R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We localized the structural domain within the p21 molecule recognized by the Y13-259 monoclonal antibody. By analysis of a series of bacterially expressed p21 deletion mutants, the monoclonal antibody was found to interact with a region between positions 70 and 89 in the p21 amino acid sequence. By comparison of the coding sequences of different p21 proteins recognized by this monoclonal antibody, a highly conserved amino acid region between positions 70 and 81 was found to be the most likely site for the epitope detected by the Y13-259 antibody. This monoclonal antibody was further shown not to interfere directly with in vitro biochemical functions of the molecule, including GTP binding, GTPase, and autokinase activities.     

Immunohistochemical analysis of normal and mutated ras oncogene p21 expression in human pulmonary and pleural neoplasms

In this study we examined 214 cases of primary human pulmonary neoplasms for the expression of a mutated form of the ras oncogene p21 product, recognized by the monoclonal antibody (MCA) DWP. Adjacent serial sections from these same cases had previously been used to demonstrate the frequency of ras p21 expression using the broadly reactive anti-ras p21 MCA RAP-5. Confirmation of the increased expression of p21 was accomplished using MCA Y13-259. The use of adjacent tissue sections from these cases allows the direct comparison of the expression of the mutated and non-mutated forms of ras p21. If reactivity with DWP would prove to be significantly more restrictive than that of the "pan" ras MCAs, RAP-5 and Y13-259, it would lend support to the possibility that DWP (and similar MCAs which detect other specific mutations) could be used to define subsets of these neoplasms based on their specific ras p21 phenotype. Since one would anticipate that the valine/cysteine substitution at position 12 of the ras p21 would occur at only low frequencies in human tumors, our results with DWP are consistent with this hypothesis. As previously reported, RAP-5 reacted with a high proportion of lung tumors (100/214 or 47%). In this report, we demonstrate the selective expression of the mutation recognized by the MCA DWP in only 5% of these same tumors (13/214), and that the expression of this mutated form is not restricted to any of the conventional histological subclasses of pulmonary neoplasms.     

Tissue distribution of smg p25A, a ras p21-like GTP-binding protein, studied by use of a specific monoclonal antibody

We made a monoclonal antibody specifically recognizing smg p25A among many ras p21-like GTP-binding proteins and investigated the tissue distribution of smg p25A by use of this antibody. By immunoblot analysis, smg p25A was detected in rat brain and bovine adrenal medulla but not in bovine adrenal cortex or other rat tissues including thymus, spleen, lung, heart, liver and kidney. However, by immunocytochemical studies, smg p25A was detected not only in the synaptic areas of rat brain and the chromaffin cells of bovine adrenal medulla but also in the endocrine cells of rat pancreatic islets, the acinar cells of rat exocrine pancreas and the exocrine cells of rat submaxillary gland. These results suggest that smg p25A is involved in the regulation of secretory processes not only in synapses but also in other endocrine and exocrine secretory cells.     

Expression of epidermal keratins and filaggrin during human fetal skin development

The major structural proteins of epithelia, the keratins, and the keratin filament-associated protein, filaggrin, were analyzed in more than 50 samples of human embryonic and fetal skin by one-dimensional SDS PAGE and immunoblotting with monoclonal and polyclonal antibodies. Companion samples were examined by immunohistochemistry and electron microscopy. Based on structural characteristics of the epidermis, four periods of human epidermal development were identified. The first is the embryonic period (before 9 wk estimated gestational age), and the others are within the fetal period: stratification (9-14 wk), follicular keratinization (14-24 wk), and interfollicular keratinization (beginning at approximately 24 wk). Keratin proteins of both the acidic (AE1-reactive, type I) and the basic (AE3-reactive, type II) subfamilies were present throughout development. Keratin intermediate filaments were recognized in the tissue by electron microscopy and immunohistochemical staining. Keratins of 50 and 58 kD were present in the epidermis at all ages studied (8 wk to birth), and those of 56.5 and 67 kD were expressed at the time of stratification and increased in abundance as development proceeded. 40- and 52-kD keratins were present early in development but disappeared with keratinization. Immunohistochemical staining suggested the presence of keratins of 50 and 58 kD in basal cells, 56.5 and 67 kD in intermediate cells, and 40 and 52 kD in the periderm as well as in the basal cells between the time of stratification and birth. Filaggrin was first detected biochemically at approximately 15 wk and was localized immunohistochemically in the keratinizing cells that surround hair follicles. It was identified 8-10 wk later in the granular and cornified cell layers of keratinized interfollicular epidermis. These results demonstrate the following. An intimate relationship exists between expression of structural proteins and morphologic changes during development of the epidermis. The order of expression of individual keratins is consistent with the known expression of keratins in simple vs. stratified vs. keratinized epithelia. Expression of keratins typical of stratified epithelia (50 and 58 kD) precedes stratification, and expression of keratins typical of keratinization (56.5 and 67 kD) precedes keratinization, which suggests that their expression marks the tissue commitment to those processes. Because only keratins that have been demonstrated in various adult tissues are expressed during fetal development, we conclude that there are no "fetal" keratins per se.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localisation of the human N-ras oncogene to chromosome 1cen - p21 by in situ hybridisation.

The N-ras gene is a transforming gene isolated from a variety of human tumour cell lines and is a member of a family of related ras genes. Somatic cell hybrids have previously shown that the N-ras gene is located on chromosome 1. We have confirmed this localisation by in situ hybridisation to metaphase preparations of lymphocytes and localised the gene to the region 1cen - p21. A survey has found 47 reported cases of malignancy involving deletions in the short arm of chromosome 1. Fifteen of the 47 involved a deletion in this region.     

The bovine papillomavirus E5 oncogene can cooperate with ras: identification of p21 amino acids critical for transformation by c-rasH but not v-rasH.

We have previously used a series of insertion-deletion mutants of the mutationally activated v-rasH gene to identify several regions of the encoded protein that are dispensable for cellular transformation (B. M. Willumsen, A. G. Papageorge, H.-F. Kung, E. Bekesi, T. Robins, M. Johnsen, W. C. Vass, and D. R. Lowy, Mol. Cell. Biol. 6:2646-2654, 1986). To determine if some of these amino acids are more important for the biological activity of c-rasH, we have now tested many of the same insertion-deletion mutants in the c-rasH form for their ability to transform NIH 3T3 cells. Since the transforming activity of c-rasH is low, we have used cotransfection with the bovine papillomavirus (BPV) genome to develop a more sensitive transformation assay for c-rasH mutants. The increased sensitivity of the assay, which is seen both in focal transformation and in anchorage-independent growth, is mediated by cooperation between the BPV E5 gene and ras. E5-dependent cooperation was seen for v-rasH as well as for c-rasH, which suggests that the major effect of E5 was to increase the susceptibility of the cell to transformation to a given level of ras activity. The cooperation assay was used to test the potential importance, in c-rasH, of codons 93 to 108, 123 to 130, and 166 to 183, which were nonessential for v-rasH transformation. Relative to the respective transforming activity of wild-type c-rasH and v-rasH, mutants with lesions in codons 102 and 103 were significantly less active in their c-rasH forms than in their v-rasH forms. We conclude that a region including amino acids 102 and 103 encodes a function that is more critical to c-rasH than to v-rasH. Guanine nucleotide exchange is one function that is compatible with such a phenotype.     

Expression of Fas,p53 and AFP in development of human fetal germ cells in vitro

In the present study we employed a two-step culture system to study the expression of Fas, p53 and alpha-fetoprotein (AFP) in the development in vitro of human fetal germ cells. p53 mRNA was determined by Northern blotting, and Fas content was assessed by western blotting. RT-nested polymerase chain reaction (RT-nPCR) analysis was performed to determine the expression of AFP mRNA in different stages of fetal follicular development. Follicular cell apoptosis was evaluated by DNA fragmentation analyses (DNA ladder). The results showed that by day 7 of culture approximately one-sixth of fetal germ cells grew to class C oocytes (primary oocytes) from class B oocytes (primordial oocytes) or class A oocytes. On day 45 of culture, one-third of these primary follicles doubled in size. In the meantime, there was a high proportion apoptosis of follicular cells on days 35 or 45 of culture, as evident by a clear ladder pattern of DNA fragmentation upon electrophoretic analysis. Expression of Fas antigen and p53 mRNA increased in a time-dependent manner, while AFP mRNA was expressed on days 10 to 35, and disappeared on day 45. These results indicate that human fetal germ cells can develop in a two-step culture system and AFP may play an active role in the proliferation of these germ cells. At the late stage of follicular development in vitro, a number of follicular cells became apoptotic. Moreover, apoptosis may be the mechanism responsible for fetal germ cell regression and the Fas antigen and/or p53-mediated death pathway may be central in the induction of germ cell regression.     

甲胎蛋白对HeLa细胞N-ras、p53和p21~(ras)表达的促进作用

大量研究已证明甲胎蛋白 (alpha fetoprotein ,AFP)对肿瘤细胞的增殖具有调节作用 .为探讨AFP对细胞生长促进作用的分子机理 ,采用从人脐带血中提取的AFP作用于体外培养的HeLa细胞 ,用Northern印迹分析法分析不同作用时间时细胞N rasmRNA的表达以及用Western印迹分析法分析p5 3、p2 1ras的表达 .结果发现 ,在AFP(2 0mg L)作用后 ,HeLa细胞的N rasmRNA、p5 3蛋白质和p2 1ras蛋白质的表达量与对照组比较在 12h和 2 4h时都有明显增加 .AFP的作用均可被抗AFP单克隆抗体所拮抗 .实验结果提示 ,AFP对细胞生长的调节作用可能通过促进这些原癌基因的表达来实现 .     

In vitro antigenic reactivity of synthetic lipid A analogues as determined by monoclonal and conventional antibodies

Cross-reactivities of synthetic lipid A analogues with monoclonal and conventional antibodies against Salmonella lipid A were studied. It was shown that the in vitro antigenicity of a synthetic compound 506, beta-(1----6) D-glucosamine disaccharide 1,4'-bisphosphate, which is acylated at 2'-amino and 3'-hydroxyl groups with (R)-3-dodecanoyloxytetradecanoyl and (R)-3-tetradecanoyloxytetradecanoyl groups, respectively, and has (R)-3-hydroxytetradecanoyl groups at 2-amino and 3-hydroxyl groups, was practically indistinguishable from that of the natural E. coli lipid A preparation, and that both phosphates in positions 1 and 4' as well as ester- and amide-linked fatty acyl residues, particularly 3-acyloxyacyl group, of the glucosamine disaccharide are involved in the cross-reactivity of lipid A as important antigenic determinants.     

Distribution of isomyosin in cultured cardiac myocytes as determined by monoclonal antibodies and adenosine triphosphatase activity

The distribution of isomyosin in cardiac muscle cells in culture has been investigated with monoclonal antibodies and Ca2+-activated myosin ATPase cytochemical staining. With immunofluorescent studies using monoclonal antibodies to isomyosins V1 and V3, the cardiac myocytes grown in a serum-free and thyroxine (T4)-free medium for 7 days contained a predominant population of cells which were strongly reactive to anti-V3 antibody. A small population of myocytes in this culture exhibited weak or no reaction to anti-V3 antibody. When cultures were exposed to anti-V1 antibody, the predominant cardiac myocyte population showed little or no reactivity to this antibody, whereas a small population of the myocytes were strongly reactive. The myosin ATPase staining reaction of the positive myocyte population was significantly less pronounced than that of the V3-negative population which showed a strong reaction. The staining pattern changed dramatically after exposure of cultured myocytes to thyroid hormone for 7 days. Most of the cells were found to react strongly with anti-V1 antibody, while some cells showed little reactivity and some were not stained at all. A small number of cardiac myocytes in this culture showed little or no reactivity to anti-V1 antibody but were strongly reactive to anti-V3 antibody. The predominant anti-V1-positive myocyte population exhibited strong myosin ATPase staining as compared to a smaller V3-positive myocyte population which showed very weak staining. The cytochemical results of ATPase staining in cardiac myocytes agreed well with ATPase activity as determined on pyrophosphate gels containing isomyosin derived from cultured cardiac myocytes with or without T4. This study has demonstrated that cultured myocytes contain a small population of muscle cells which is not responsive to thyroid hormone or to the lack of it.     

3p21, 5q21, 9p21 and 17p13 allelic deletions accumulate in the dysplastic spectrum of laryngeal carcinogenesis and precede malignant transformation

A tissue field of somatic genetic alterations precede the histopathological phenotypic changes of carcinoma. Loss of Heterozygosity (LOH) at the sites of known or putative tumor suppressor genes is a common genetic abnormality detected in precancerous conditions. These genomic changes could be of potential use in the diagnosis and prognosis of pre-malignant laryngeal lesions. Recently the concept of laryngeal intraepithelial neoplasia (LIN) was introduced. To evaluate patients with an increased risk of developing invasive laryngeal carcinoma via a dysplasia-carcinoma progression we investigated 102 microdissected cell populations. Cell populations were procured from 15 laryngectomy specimens with different peritumoral histological changes adjacent to the squamous cell carcinoma cells and 15 laryngeal endoscopic biopsies with no evidence of malignant transformation in a 6-10-year follow-up period. Histological diagnoses were subdivided into keratosis without dysplasia (KWD), with mild dysplasia (LIN 1), with moderate dysplasia (LIN 2), and with severe dysplasia or carcinoma in situ (LIN 3). Microsatellite analysis was performed with the aim of studying LOH of 5q21 (APC), 9p21 (p16), 3p21 and 17p13 (p53) chromosomal regions. Frequent allelic losses were found in carcinoma cells at p53 (54%), p16 (66%), 3p21(87%) and 5q21(58%). Identical LOH patterns were determined in 100% of the LIN3 peritumoral cells, 60% of LIN2, 50% of LIN 1 and 25% of KWD. In contrast, histologically normal mucosae, KWD and LIN1 lesions without malignant progression showed no allelic loss. These results show that dysplasia correlates with LOH at 3p21, 5q21, 9p21 and 17p13 in early laryngeal carcinogenesis. These genomic changes in pre-malignant laryngeal lesions could be of potential use as markers for cancer risk assessment.     

Expression of the human ETS-2 oncogene in normal fetal tissues and in the brain of a fetus with trisomy 21

Summary The expression of the ETS-2 proto-oncogene, located on chromosome 21, in normal fetal tissues and in neural tissue of a fetus affected by Down syndrome has been investigated. The results show that the ETS-2 proto-oncogene is expressed in almost all the tissues examined and that it is transcribed at constant levels in neural tissue between the 13th and 24th weeks. ETS-2 experession appeared to be slightly increased in Down syndrome brain compared with that of normal controls of the same gestational age.     

Expression of fibroblast growth factors 18 and 23 during human embryonic and fetal development

Fibroblast Growth Factor (FGF) 18 and 23 are two recently identified members of the FGF family, a family of structurally related polypeptides with diverse roles in physiological and pathological processes. Studies mostly performed in rodents and chicken have demonstrated that FGF18 is a pleiotropic growth factor involved in the development of various organs, while there are no data supporting a direct role of FGF23 in cell proliferation or differentiation either in physiology or pathology in any species. However, it is now established that FGF23 can be a humoral messenger and an important regulator of phosphate homeostasis and vitamin D metabolism. As a first step towards elucidating the roles of these FGF in human development, we examined FGF18 and FGF23 mRNA expression by in situ hybridization in whole human embryos at 30 days and 8 weeks of gestation (GW) and in specific fetal tissues at different ages. We report a highly restricted expression pattern for both FGF genes in human embryonic development.     

Expression of viral p21ras during acquisition of a transformed phenotype by rat adrenal cortex cells infected with Kirsten murine sarcoma virus.

Rat adrenal cortex cells infected with Kirsten murine sarcoma virus acquire a transformed phenotype in a progressive fashion. The expression of the viral p21ras does not appear to correlate with the degree of transformation of the adrenocortical cells but rather is produced at similar levels as the culture becomes transformed. This indicates that the expression of an oncogenic form of p21ras is not of itself sufficient to completely transform rat adrenal cortex cells.