Diversity of Cellular Molecules in Human Cells Detected by Monoclonal Antibodies Reactive with c-myc Proteins Produced in Escherichia coli

Six clones of monoclonal antibodies, MYC-1 to -6, were prepared by using two kinds of truncated c- myc proteins, p23 and p42, produced in Escherichia coli as immunogens. Analysis with enzyme-linked immunosorbent assays and immunoblotting assays with peptides produced in Escherichia coli showed that 5 clones of monoclonal antibodies, MYC-1 to -4 and -6, were reactive with c- myc protein encoded by exon 2. The remaining one clone, MYC-5, was reactive with the portion of c- myc protein encoded by exon 3. All monoclonal antibodies were also reactive with phosphorylated c- myc protein produced by insect cells infected by the baculovirus expression vector with the human c- myc gene. With immunoblotting assays using cellular lysates, MYC-1 and -3 detected bands at the levels of 58 kDa and 60 kDa, MYC-5 detected a band at 56 kDa and MYC-6 detected bands at 68 kDa and 75 kDa. All of these bands were detectable in nuclear extracts of HL-60 and Colo320, both of which have amplified c- myc genes, and also the extract of RmycYl which is the c- myc gene transfectant into 3Y1 rat cells. None of them was detectable in peripheral blood mononuclear cells and 3Y1, both of which lacked activated c- myc genes. This indicates that these nuclear proteins are either c- myc gene products or molecules closely related to the c- myc gene. The remaining two clones, MYC-2 and -4, detected a band at the level of 85 kDa in cytoplasmic extracts of all the above-mentioned cells independent of the presence of the c-myc gene. This suggests that 85 kDa protein might be irrelevant to the c- myc gene. The 56 kDa protein was detectable by MYC-5 in phytohemagglutinin-stimulated peripheral blood mononuclear cells as well as leukemic cells of some patients.     

Immunohistochemical detection of c-myc products in colorectal cancer and proliferative cell rate.

Expression of c-myc protein was studied immunohistochemically in colorectal cancers using a monoclonal antibody, MYC-1. Immunoblotting assays with cellular lysates demonstrated a band of the gene products at the level of 60 kDa. c-myc-protein-positive tumor cells were observed in 43 (43.4%) of 99 specimens of colorectal cancers. There was no significant correlation between the incidence of MYC-1-positive tumors and clinicopathological findings. The rate of MYC-1 proteins occurring in patients with liver metastasis was significantly higher than that in patients without the metastasis. The rate of occurrence of DNA polymerase-alpha-positive cells in MYC-1 protein-positive tumors was significantly higher than in MYC-1 negative ones. The results suggested that MYC-1 immunoreactivity might possibly be a useful prognostic marker of colorectal cancers.     

Expression of c-myc oncogene product and ras family oncogene products in various human malignant lymphomas defined by immunohistochemical techniques

The authors studied the expression of c-myc and ras family oncogene products in 43 cases of malignant lymphoma (ML) using the immunoperoxidase method. Unfixed frozen sections of lymph nodes from four patients with Hodgkin's disease and 39 with non-Hodgkin's lymphoma, together with normal lymph nodes, were studied by the avidin-biotin-peroxidase complex (ABC) technique. Two monoclonal antibodies, MYC-2 raised against recombinant human c-myc protein (reacting specifically with the c-myc products P62 and P67) and RASK-4 (raised against recombinant P21 and reacting specifically with ras-family product P21) were used. The c-myc product was detected in nuclei of ML cells and some normal, mainly germinal center, lymphocytes. When the staining intensity shown by normal germinal-center lymphocytes was graded as positive (+) or weakly positive (+/-), a very intensely positive reaction ( to ++) was observed in 37 cases (86%) of ML, a positive reaction (+) in four cases (9.3%), and a weakly positive reaction (+/-) in two cases (4.7%). The ras family oncogene product reaction was intensely positive (++) in two cases (4.7%), positive (+) in 16 cases (37.2%), weakly positive (+/-) in 13 cases (30.2%), and negative in 12 cases (27.9%). Western blot analysis confirmed an elevated level of c-myc products in two cases, which showed intense MYC-2 staining, and of ras family products in one case, which demonstrated intense RASK-4 staining. The enhanced expression of these gene products may play an important role in lymphomagenesis of such cases.     

Correlation of c-myc expression with nuclear pleomorphism in human renal cell carcinoma

The expression of the c-myc gene product in renal cell carcinomas was examined by immunostaining with monoclonal antibody (mAb) MYC-1. The effects of preservation and fixation of tissues on staining were first examined. In cryostat sections fixed with 4% buffered formalin for 15 min, staining was observed in the nucleus. On the other hand, in paraffin sections after fixation with 10% formalin, staining was observed in the cytoplasm, but not in the nucleus. Because c-myc protein has been shown to be a nuclear protein, the finding that c-myc protein was not detectable in the nucleus appeared to be due to the preservation or fixation procedures used. Therefore, cryostat sections fixed with 4% formalin were used to investigate the correlation between the reaction of MYC-1 mAb and nuclear pleomorphism in primary and metastatic renal cell carcinomas. Among 41 primary tumors, positive staining was observed in 2 of 17 tumors (12%) of grade 1, 17 of 21 (81%) of grade 2, and all 3 (100%) of grade 3. Among 17 metastatic tumors, positive staining was not observed in any of the 5 (0%) of grade 1 but was observed in 2 of 4 (50%) of grade 2 and all 8 (100%) of grade 3. Thus, the frequency of the positive reaction with MYC-1 mAb was correlated with nuclear pleomorphism in primary and metastatic renal cell carcinomas. The reaction of Ki-67 mAb, which recognized a nuclear antigen present in proliferating cells, was also correlated with nuclear pleomorphism. These findings suggest that the c-myc gene product plays a role in cell proliferation in renal cell carcinomas.     

Evolutionary conservation of the product of human c-myc exon 1 and its inducible expression in a murine cell line

The human c-myc proto-oncogene contains an open reading frame within its first exon which is translated into protein (MYCHEX1). While the murine c-myc exon 1 is obviously non coding, we show that in mouse cells there are polypeptides closely related to human MYCHEX1. These polypeptides share the same immunological reactivities with the human polypeptides. Furthermore, the 32 kDa polypeptide of murine cells has, like its human counterpart, the ability to dimerise in a 58 kDa form in denaturing and reducing SDS-PAGE. The human gene was introduced into a murine cell line by transfection. A cell line was studied, in which the inducible expression of the gene allows a substantial increase in the concentration of the corresponding protein. This inducible protein behaves in any respect like the murine one, either in SDS-PAGE or in a specific immunoassay. These shared properties constitute a further proof that the human and mouse MYCHEX1 proteins are encoded by the sequence overlapping the human myc exon 1 and a related murine sequence. The gene contained in the human c-myc exon 1 is not, therefore, a specific feature of human cells.     

Characterization of bcr gene products in hematopoietic cells

The bcr gene plays a critical role in the pathogenesis of two human leukemias associated with the Philadelphia chromosome: chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL). In both instances a chimeric gene, formed between bcr and the abl protooncogene, results in expression of fused bcr-abl proteins with tyrosine kinase activity. There is controversy regarding the normal gene products of bcr. We investigated this problem by several techniques and found proteins of 190/185, 155, 135, 125, 108, 83 and 47 kDa in several human cell lines by immunoprecipitation with two distinct site-directed anti-bcr antibodies termed anti-bcr(738-753) and anti-bcr(898-911). The 190/185, 155, 125 and 108 kDa proteins were consistently detected by anti-bcr(898-911) antibodies by immunoblotting. Antibodies pre-reacted with excess bcr peptide did not detect these proteins. These proteins were labeled with [32P]orthophosphate in vivo and in vitro by [gamma 32P]ATP in immune complex kinase assays performed with anti-bcr antibodies indicating that these proteins are phosphorproteins. Following labeling in kinase assays, phosphoamino acid analyses detected both phosphoserine and phosphothreonine. In structural studies using one dimensional peptide maps derived by partial V8 protease treatment, the 185, 155, 135, 125 and 108 kDa proteins shared several peptide fragments but contained unique fragments as well. Similarly 2-dimensional maps of proteins labeled in the kinase assay exhaustively digested with trypsin, revealed homology between the 155, 135, 125, 108, and 83 kDa proteins. bcr proteins sedimented in glycerol gradients as putative complexes detected in the cytoplasm of the cell. A 47 kDa protein as well as the recently identified Ph-P53 protein appeared to be associated with bcr proteins based on their co-sedimentation in glycerol gradients and co-immunoprecipitation with several different anti-bcr antibodies. None of the proteins exhibited a precursor-product relationship in pulse-chase experiments conducted with [35S]methionine. We conclude that human cells express several different bcr gene products ranging in size from 190 to 83 kDa, and that these proteins can form specific intracellular cytoplasmic complexes with other cellular proteins.     

Expression of c-myc gene product in gastric carcinoma

The expression of c-myc oncogene product was studied in 213 cases with gastric carcinoma by an immunoperoxidase method using a monoclonal antibody (MYC-1). Fifty (23.5%) of 213 tumors showed immunoreactivity to MYC-1. The distribution of c-myc-product-positive cells was observed mainly at the marginal area of the tumor. Excess reactivity to c-myc product occurred more frequently in invasive cancers than in localized cancers, and c-myc production expression in cancer tissue correlated well with peritoneal dissemination. Patients with c-myc-protein-positive tumor had significantly poorer prognosis than those with c-myc-protein-negative tumor in invasive gastric carcinomas, and the c-myc product status correlated well with the recurrence of cancer by peritoneal dissemination. These results suggest that the expression of c-myc gene product might be related to the proliferative activity of gastric carcinoma and serve as a new biologically relevant tumor marker for determining the prognosis.     

Binding of transcription factor sp1 to exon-1 of C-myc is altered in burkitts-lymphoma

In Burkitt's lymphoma (BL) a consistent chromosomal translocation involving the c-myc proto-oncogene locus on chromosome 8 and one of the immunoglobulin loci located on chromosomes 14, 22 or 2 results in the aberrant expression of c-myc. In six out of ten published BL sequences the translocated c-myc gene contains mutations and/or deletions in an Spl site in the first exon. We show by competition gel mobility shift assays and association constant comparison that the binding of Spl to this site is altered in four of these clones. The affinity of Spl for this site is increased 5-10 fold in Burkitt's lymphoma BL22 and decreased approximately 5 fold in BL2. Mutations at this site in cell lines Raji and Daudi also show a decrease in Sp1 binding when compared with the wild type sequence.     

Clonal cosegregation of tumorigenicity with overexpression of c-myc and transforming growth factor alpha genes in chemically transformed rat liver epithelial cells.

Tumorigenicity was correlated with levels of expression of the genes for transforming growth factor alpha (TGF-alpha), epidermal growth factor receptor, c-myc, c-H-ras, and c-K-ras in a series of 16 clonally derived transformed liver epithelial cell lines. The clonal lines, which varied in tumorigenicity from 0 to 97%, were established from a phenotypically heterogeneous population produced by repeated exposure of diploid WB-F344 (WB) cells to N-methyl-N'-nitro-N-nitrosoguanidine. Segregation of gene expression with tumorigenicity among clonal lines was determined by correlating rank orders of gene expression by clones relative to expression by wild-type WB cells. Only the expression of the c-myc gene correlated with tumorigenicity among all transformed clones. TGF-alpha gene expression was not correlated with tumorigenicity among all clones, but it was highly correlated with tumorigenicity among clones that expressed the c-myc gene above the median level for all clones (greater than 5-fold the level of expression by WB cells). Even high levels of expression of the TGF-alpha gene (up to 60-fold the level of expression by WB cells) were not correlated with tumorigenicity among the clones expressing the c-myc gene at levels less than 5-fold the level of expression by WB cells. Clones which simultaneously overexpressed both c-myc and TGF-alpha genes at levels above the median levels for all clones were significantly more tumorigenic than were clones which expressed either or both genes at lower than median levels. These results suggest that overexpressed c-myc and TGF-alpha genes cooperate in their association with tumorigenicity. Most of the highly tumorigenic clones that overexpressed c-myc and TGF-alpha also overexpressed the c-H-ras and/or the c-K-ras genes; clones that overexpressed neither of the c-ras genes nor the genes for c-myc and TGF-alpha were not very tumorigenic, while clones that expressed one or both c-ras genes (but not both c-myc and TGF-alpha) were variably tumorigenic over an intermediate range.     

Preparation of antigens from Trichophyton mentagrophytes using a new semi-solid culture medium and their characterization by SDS-PAGE and immunological techniques.

A new semi-solid culture medium was developed by substituting the agar in Sabouraud glucose medium by Lutrol FC 127 (BASF, Ludwigshafen, Germany). This culture medium can be liquefied by cooling it down from the incubation temperature to 5-10 degrees C, thus allowing the full harvest of fungal mycelium without any contamination by the gelling agent for antigen preparation. More than 25 protein bands with molecular weights in the range of 98 to 12 kDa were fractionated by SDS-PAGE in antigen preparations from Trichophyton mentagrophytes. Hyperimmune antisera were raised in rabbits and used for immunological studies. Heat-inactivated mycelium was used for the absorption of antibodies against heat-stable cell wall constituents. This absorption facilitated the detection of specific protein bands during immunoblotting which revealed 17 protein antigen bands reacting with antibodies over a range of molecular weights from 98 to 24 kDa.     

Nucleotide sequence of the rat Bmyc gene

We have cloned and sequenced the rat Bmyc gene. The rat Bmyc gene contains sequences related to the central part of c-myc, namely the first intron, the second exon, and the noncoding part of the third exon. The homology drops in the 3' part of the c-myc second exon, but continues in the noncoding part of the third exon. We have sequenced the total predicted coding region of the Bmyc. The longest open reading frame in Bmyc suggests a protein of 178 amino acids, which is only 41% of the c-myc protein size. To confirm the putative open reading frame, we have produced a trpE-Bmyc protein that is detected with a pan-myc antibody. We discuss these findings in the context of potential functional domains and the possibility of overlapping and distinct activities of myc-family proteins.     

脂肪肉瘤c-myc和p53基因蛋白的表达及意义

背景与目的：原癌基因c-myc和抑癌基因p53与脂肪肉瘤的关系的研究较少,脂肪肉瘤中是否存在p53基因突变文献报道不一,本文拟探讨脂肪肉瘤中c-myc、p53基因蛋白表达的水平及意义,以期为本组肿瘤发生发展及病理诊断、鉴别诊断提供一定的分子生物学资料。方法：采用链菌素抗生物素－生物素（labelled streptavidin-biotin,LSAB)免疫组化法、聚合酶链反应－单链构象多态性分析（Single strand conformation polymorphism analysis of polymerase chain reaction products,PCR-SSCP)及DNA序列分析方法。结果：c-myc和p53蛋白在脂肪肉瘤表达阳性率分别为38．09％（16／42）和48．08％（25／52）。不同类型脂肪肉瘤,分化良好者阳性率均明显低于分化较差者。脂肪肉瘤中c-myc和p53蛋白表达呈正相关。c-myc和p53蛋白表达在原发者和复发者之间的差异均无统计学意义。p53第6、7、8外显子PCR－SSCP分析,2例多形性脂肪肉瘤出现异常泳动带。DNA序列分析证实1例第8外显子第268位编码区出现错义突变（AAC→ATC）,另1例第6例显子第221位编码区出现可疑杂合性同义突变（GAG→GAA)。结论：c-myc和p53蛋白与脂肪肉瘤的形成及分化和恶性程度有关。它们可作为判断脂肪肉瘤分化程度及恶性程度参考指标之一,但与肿瘤复发无关。在脂肪肉瘤发生发展中两者可能起协同作用。脂肪肉瘤中p53基因第6、8外显子分别存在点突变。     

Use of the OM-11-906 monoclonal antibody for determining p62 c-myc expression by flow cytometry in relation to prognosis in colorectal cancer

The expression of the c-myc protein product (p62 c-myc) and deoxyribonucleic acid (DNA) ploidy status was determined by a flow cytometric technique in 83 patients with colorectal cancer followed up for a median of 30 months (range 6-60 months). The OM-11-906 antibody, used to detect p62 c-myc, revealed a 62 kDa and 45 kDa band on Western blots in tumours. Correlation of quantitative dot blotting of tumour mRNA to flow cytometric p62 c-myc expression was good (r = 0.87, P less than 0.01). Levels of p62 c-myc varied in colorectal cancer and low levels (less than 20 fluorescein units) correlated with improved survival (log rank chi 2 = 4.69, df = 1, P = 0.03), and this was a better prognostic index than DNA ploidy (log rank analysis chi 2 = 2.38, df = 1, P less than 0.1). Although expression of the c-myc gene was found, using the OM-11-906 antibody, to be a prognostic feature in colorectal cancer, these and other results need to be interpreted with caution given the presence of two protein bands by Western blotting.     

Preparation of anti-ras Mr 21,000 protein monoclonal antibodies and immunohistochemical analyses on expression of ras genes in human stomach and thyroid cancers

Sixteen clones (RASK-1 to -16) of murine monoclonal antibodies were raised against ras Mr 21,000 protein (p21). The p21 produced by Escherichia coli with inserted v-Ki-ras genes was used as immunogen. RASK-1 was found to be specific for Ki-ras p21, whereas RASK-2 to -16 reacted with the p21s of Ki-, N-, and Ha-ras genes in both enzyme-linked immunosorbent and immunoblotting assays. Binding inhibition assays with biotinylated monoclonal antibodies by enzyme-linked immunosorbent assay showed that the monoclonal antibodies of the 16 clones included those binding to several mutually distinct sites on p21. The expressions of ras p21 in human stomach and thyroid tissues were examined with RASK-3, which reacted with all the Ki-, N-, and Ha-ras p21s immunohistochemically by the avidin-biotin peroxidase complex method. Formalin-fixed, paraffin-embedded tissues of 101 cases of stomach cancer, 53 cases of noncancerous stomach, 74 cases of cancer of the thyroid, and 59 cases of noncancerous thyroid were analyzed. In both the stomach and thyroid, cancer cells expressed p21 predominantly. Cells of cases with various noncancerous disorders as well as certain types of normal cells were also p21 positive. These findings suggest that precaution is required in use of p21 as a cancer marker. Expression of p21 was noted in moderately to well-differentiated stomach cancer, intestinal metaplasia, and atypical hyperplasia. This finding suggests that the appearance of p21 in stomach cancer may be initiated before cytological transformation.     

Identification of the ret proto-oncogene products in neuroblastoma and leukemia cells

Monoclonal and/or polyclonal antibodies were generated against the products synthesized from two portions of the ret proto-oncogene (c-ret) cDNA expressed in Escherichia coli. These antibodies were reactive in immunoblotting with 150 kd and 170 kd proteins in cell lysates from three human neuroblastoma cell lines expressing the ret proto-oncogene. When the neuroblastoma cells were treated with tunicamycin, a protein with an apparent molecular weight of 120 kd, which is consistent with that of the c-ret protein predicted from the cDNA sequence, appeared on immunoblots. These results indicated that the 150 kd and 170 kd proteins in neuroblastoma cells are produced from a single polypeptide of 120 kd by posttranslational glycosylation. Furthermore, the antibodies detected a unique 190 kd protein as well as 150 kd protein in a cell lysate from THP-1 human monocytic leukemia cell line, suggesting that glycosylated forms of the c-ret protein are different between neuroblastoma and leukemia cells.     

Identification and characterization of a rat protein (p 105) auto-antigenic in rats bearing a progressive syngeneic colon carcinoma.

Sera from BDIX rats inoculated with 2 tumor clones derived from a single syngeneic colon carcinoma were assayed by Western blotting for the presence of antibodies against the grafted tumor. The PROb clone is progressive and produces metastases. We observed that rats bearing this tumor developed antibodies against an unglycosylated water-soluble protein of 105 kDa. The magnitude of this humoral response, as assessed by the intensity of the signal on immunoblots, was inversely correlated with survival of the rats. Furthermore, rats inoculated with the REGb clone, which is immunologically rejected, never developed detectable antibodies against the tumor. Antisera from rats injected with PROb tumor detected p105 antigen in cellular extracts from the REGb clone and from a series of rat and human cell lines. This protein was also detected in variable amounts in some normal adult and fetal tissues. Treatment of PROb or REGb cells by either interferon-gamma or heat shock did not significantly alter the expression of the p105 auto-antigen.