Chromaffin granule and PC12 cell chondroitin sulfate proteoglycans and their relation to chromogranin A.

Two major proteoglycans, which appear to be structurally closely related, were isolated from bovine chromaffin granule matrix proteins by ion-exchange chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis they have apparent average molecular sizes of 35-40 kDa (range of 23-75 kDa) and generate a 14-kDa core glycoprotein after chondroitinase treatment. Previous studies demonstrated that these two major chromaffin granule proteoglycans are very similar in terms of their peptide mapping patterns and carbohydrate composition (having a high proportion of tri- and tetraantennary N-glycosidic oligosaccharides, and O-glycosidic oligosaccharides consisting predominantly of disialyl derivatives of galactosyl(beta 1-3)N-acetylgalactosamine), and that they differed in these respects from the chromogranins. By using antisera to five synthetic peptide fragments of chromogranin A to stain immunoblots of purified chromaffin granule proteoglycans before and after chondroitinase treatment, we have now shown that these major proteoglycans are not immunochemically related to chromogranin A. However, it has recently been reported that some chromogranin A-immunoreactive material disappears after chondroitinase treatment, and our studies demonstrate that approximately 1-2% of the chromogranin A occurs in the form of a 110-kDa proteoglycan, which is converted to a 95-kDa core glycoprotein after chondroitinase treatment. Similar chromogranin A proteoglycans could be detected in rat PC12 pheochromocytoma cells, where they have a molecular size of 115-145 kDa and yield a 105-kDa core protein after chondroitinase treatment. Studies using antibodies to synthetic peptide fragments of chromogranin B (secretogranin I) did not provide any evidence that this related protein occurs in a proteoglycan form.

ABO blood type predicts the cytolocalization of anti-P-glycoprotein monoclonal antibody reactivity in human colon and ureter.

Classic multidrug resistance is mediated by a P-glycoprotein. Using monoclonal antibody C219 (MAb C219) in an immunohistochemical study, we found high levels of putative Golgi P-glycoprotein in normal columnar and transitional epithelium in subpopulations of patients with specific blood types. For example, Golgi staining was present in blood type A patients in 46% of normal colon samples (N = 21) and 88% of normal ureter samples (N = 17). In comparison, Golgi staining was present in blood group O patients in only 6% of normal colon samples (N = 34) and in 0% of normal ureter samples (N = 19). The association of MAb C219 Golgi staining with blood type A and lack of Golgi staining with blood type O was statistically significant in normal colon (P = .001) and normal ureter (P less than .0001). Inappropriate hyperexpression of P-glycoprotein was frequently found in colon carcinomas. Additional evidence that Golgi MAb C219 reactivity represents P-glycoprotein is presented. This includes (1) immunostaining of Golgi with two anti-P-glycoprotein MAbs, C219 and JSB-1, and (2) experiments in which Mab C219 Golgi reactivity was blocked by preincubation of MAb C219 with a specific P-glycoprotein epitope-containing peptide. The high degree of association of Golgi P-glycoprotein with blood type A may suggest a role for P-glycoprotein in processing or trafficking of specific blood group antigens.

Gangliosides in the human glioma cell line U-118 MG grown in culture or as xenografts in nude rats.

This study was undertaken to characterize gangliosides in the human glioma cell line U-118 MG. The cell line was grown both in cell culture and as xenografts in nude rats. A common finding in both culture and xenograft cells was the high proportion of the lactoseries ganglioside 3'-LM1, approximately one third of the total ganglioside sialic acid. Otherwise, there were marked differences between the two cell sources. The cells grown in culture had a more simple ganglioside pattern than those grown in xenografts. In the latter instance, more complex gangliosides of the lactoseries, including 3'8'-LD1, sialyllactonorhexaosylceramide and a branched structure with two terminal NeuAc alpha 2-3Gal beta 1- 4GlcNAc chains, and the gangliotetraose series were found. Another marked difference involved GM2, which in the cultured cells was a major fraction, indicating that the synthesis of the gangliotetraose series gangliosides in the former stopped at the level of GM2. These results show that the ganglioside composition of a glioma cell line is strongly influenced by environmental factors.

Metabolism of low-density lipoprotein in differentiated and undifferentiated HT29 colon cancer cells.

The metabolism of human low-density lipoproteins was studied in 2 subpopulations deriving from cells of HT29, a human colon carcinoma cell line. When grown on standard medium (25 mM glucose), about 95% of these cells are undifferentiated (G+ cells). From this heterogeneous population, a subpopulation with features of differentiated small-intestinal cells was selected by glucose deprivation (G- cells). The characteristics of the LDL receptor were first investigated. The results showed that the binding of 125I-LDL to G+ and G- cells performed at 4 degrees C was saturable and specific. The Kd values were not statistically different in the 2 cell subpopulations. The Bmax of G+ cells was 55 +/- 6 ng 125I-LDL/mg cell protein and showed no changes whatever the phase of culture. In G- cells, the Bmax was higher during the exponential phase of culture and decreased in the post-confluent phase (82 +/- 5 versus 15 +/- 6.8 ng 125I-LDL/mg cell protein). Cellular degradation of 125I-LDL was effective in both cell subpopulations but time-course studies showed that, in post-confluent G- cells, degradation was slowed as compared to G+ cells (4 hr vs. 2 hr to reach maximal degradation). The rate of LDL processing at 37 degrees C was enhanced by pre-incubation with FCS-supplemented medium, suggesting the existence of a serum component which stimulates the total degradation of 125I-LDL. Concerning regulation of the LDL receptor activity, we demonstrated that pre-incubation of G+ cells with LDL induced 80% down-regulation of receptor number in both phases of culture. This was also observed in G- cells during the exponential phase while only a 20% decrease of the receptor number was observed in post-confluent G- cells. The LDL degradation of G+ cells resulted in an inhibition of the cholesterogenic activity by 30% and 60% depending on the phase of culture. In G- cells, LDL pre-incubation inhibited cholesterol synthesis to the same extent (45%) in the exponential phase but did not affect the rate of cholesterol synthesis when cells were confluent. The defective regulatory role of LDL on receptor number and cholesterol synthesis suggests that, in the post-confluent differentiated cells, cholesterol derived from LDL does not reach the regulatory pool. Taken together, our findings indicate the existence of functional LDL receptors in the HT29 cell line, either in the differentiated or in the undifferentiated form.

Analysis of human tumor associated Thomsen-Friedenreich antigen.

The Thomsen-Friedenrich (TF) antigen is a precursor structure of MN blood group antigens and is also expressed by about 90% of human carcinomas. The immunodominant group of TF antigen [beta-galactosyl(1-3)-alpha-N-acetylglactosamine] is present in cryptic form in normal RBC and is revealed by neuraminidase treatment. A murine monoclonal antibody (Mab 49H.8) developed against neuraminidase treated human RBC was reactive against a variety of human tumors. We have characterized the human tumor associated TF antigen detected by this antibody from a human transitional bladder carcinoma cell line (647V), a human colon adenocarcinoma cell line (LS174T), and a pleural effusion fluid of a breast adenocarcinoma patient (PE 89). A heterologous sandwich radioimmunoassay for TF antigen was developed using Mab 49H.8 as the catcher and 125I-peanut agglutinin as the probe. Detergent extracts of 647V and LS174T cells, media conditioned by culturing these cells, and PE 89 were shown to contain the antigen by this assay. The specificity of the antigen capture by Mab 49H.8 in this assay was demonstrated by its selective inhibition by nitrophenyl-beta-D-galactoside, phenyl-beta-D-galactoside, and a TF hapten. Preliminary studies on TF antigen in serum samples using this assay showed that about 53.7% of the carcinoma samples contained an antigen concentration greater than 200 units/ml whereas for 90.9% of the normal samples the antigen concentration was below 200 units/ml. These studies demonstrated that the TF antigen is shed by the tumor cells both in vitro and in vivo. The TF antigen was sensitive to treatment with alkali (0.1 M NaOH for 5 h at 37 degrees C) and periodate (10 mM sodium periodate for 1 h at room temperature), was resistant to acidic pH (50 mM acetate buffer, pH 4.5, for 5 h at 37 degrees C), and could be extracted with perchloric acid (0.6 M for 1 h at 4 degrees C). The antigen was shown to be a high molecular weight glycoprotein (Mr greater than 1,000,000) by gel filtration chromatography. The density of the antigen was estimated to be about 1.35 g/ml by cesium chloride density gradient centrifugation. The antigen could be isolated from conditioned media by a combination of affinity chromatography and gel filtration with an overall purification of about 61,432-fold and a final recovery of 53.2%.(ABSTRACT TRUNCATED AT 400 WORDS)

Recognition of N-glycosidic carbohydrates on esophageal carcinoma cells by macrophage cell line THP-1.

Cell-to-cell contact between macrophages and tumor cells is an important initial reaction in a host defense mechanism against tumor cells. The authors have studied cell surface components of human esophageal carcinoma cells recognized by macrophages. Superoxide release from THP-1 cells, a human macrophage cell line, was analyzed in their interaction with a battery of human squamous cell carcinoma cell lines (TE) originated from esophageal cancer patients. The macrophage-triggering ability of TE 1 cell line, a high stimulant, was reduced after treatment with trypsin or tunicamycin, an inhibitor of N-glycosidic glycosylation. Addition of monosaccharides was efficient in competitive inhibition of these cellular interaction. Moreover, con-A-resistant mutation of TE 1 cells was found to reduce their macrophage-triggering ability, associated with increase of L-PHA-binding capacity, suggesting substitution to the GlcNAc beta(1----6)-linked lactosamine antenna in N-glycosidic carbohydrates. These findings suggest that terminal residues of N-glycosidic carbohydrates on some esophageal carcinoma cells may contribute to the recognition sites of macrophages.

Isolation of an amplified DNA sequence in stomach cancer.

By use of the in-gel DNA renaturation method, the presence of amplified DNA sequences was demonstrated in KATO-III, a cell line established from a signet ring cell carcinoma of the stomach. A DNA fragment from one of these amplified regions in KATO-III cells was cloned and designated SAM0.2; the locus containing the SAM0.2 fragment was referred to as SAM. The SAM locus was shown to be amplified not only in KATO-III cells, but also in three of 24 surgical specimens of stomach cancers and in two of 13 xenografts of human stomach cancers, all of these specimens being poorly differentiated adenocarcinoma or mucinous adenocarcinoma of the stomach. The SAM locus was not amplified in 14 cell lines of cancers of other organs or in 42 surgical specimens of lung cancers.

Characterization, in some human breast cancer cell lines, of gastrin-releasing peptide-like receptors which are absent in normal breast epithelial cells.

The binding of 125I-Tyr4 bombesin was investigated on plasma membranes of 8 human breast cancer cell lines and 2 long-term cultures of normal human breast epithelial cells. Scatchard plots were compatible with high-affinity, single-site class of receptors in 3 cell lines (KD of 0.75 x 10(-9) and 10(-9) M, Bmax of 0.75 x 10(-13) and 9.7 x 10(-13) M/mg protein in MDA-MB231 and in T47D cells, respectively) while no binding was observed in 5 other cell lines and normal epithelial cells. The neuropeptide and its structural analogues (natural or synthetic) inhibited the binding of 125I-Tyr4 bombesin in the following order of potency: gastrin-releasing peptide (GRP, EC50 = 1.7 x 10(-10) M) greater than BIM 26159 greater than bombesin, Tyr4 bombesin greater than BIM 26147 greater than litorin greater than neuromedin C. In contrast, 125I-Tyr4 bombesin binding was not displaced by neuromedin B, somatostatin, bradykinin and insulin. In agreement with our binding data, SDS-PAGE of the complex 125I-Tyr4 bombesin-receptor covalently linked by ethylene glycol-bis succinimidyl succinate (EGS) identified after autoradiography a single band with a molecular weight of 75,000, which disappeared in the presence of bombesin in excess. No transcription of either GRP or neuromedin B mRNA could be shown in tumor or normal cells. Exogenous gastrin-releasing peptide had no effect on growth of the cell lines when a serum-free medium was used, implicating that in breast cancer cell lines this receptor does not mediate growth but has a functional role.

Radioligand binding characteristics of beta 2-adrenoceptors of cultured melanoma cells.

The existence of beta-adrenoceptors on intact cells of the human malignant melanoma cell line A-375 was investigated using the binding properties of the tritiated radioligand (-)-[3H]CGP-12177, a hydrophilic non-selective beta-adrenoceptor antagonist. Displacement experiments of the radioligand from its binding site were performed with antagonists and agonists to determine the beta-adrenoceptor subtype selectivity. The binding of (-)-[3H]CGP-12177 was saturable, of high affinity (KD = 0.025 nmol/l, n = 12) and was rapid and readily reversible. The maximal number of binding sites (Bmax) was 33.5 +/- 1.9 fmol/10(7) cells or 2018 +/- 114 receptors per cell. beta-adrenoceptor antagonists inhibited binding of the radioligand with monophasic displacement curves. IC50 values were (nmol/l): propranolol (non-selective) 2.82, alprenolol (non-selective) 2.0, ICI 118,551 (beta 2-selective) 3.5 and bisoprolol (beta 1-selective) 2200. Agonists inhibited binding in the order of potency of isoprenaline greater than adrenaline greater than noradrenaline. It is concluded that cells of the melanoma cell line A-375 contain a homogeneous population of beta 2-adrenoceptors.

Cytokeratin expression during AFB1-induced carcinogenesis.

The early stages of the carcinogenic process induced by aflatoxin B1 (AFB1) in rat liver during 24 weeks of feeding and the resulting tumours have been studied with respect to cytokeratin (CK) expression. A previously uncharacterized monoclonal antibody, MRCTU/J1, has been shown to recognize rat CK18 and together with antibodies against human CK8, 18 and 19, has been used to examine the possible lineage of tumour cells and also to identify the altered foci that might be most relevant to tumorigenesis. Results suggested that AFB1-induced transformation in liver may occur in more than one cell type, since tumours with the normal hepatocyte CK pattern and those with bile duct or oval cell CK phenotype were identified. Additionally, hepatocytes with a bile duct CK phenotype appeared during the early stages of carcinogenesis. The in vivo pattern of CK expression also appeared to be maintained in one normal and one hepatoma-derived cell line. Overexpression of CKs (particularly of CK19) was a much more selective marker for altered foci, compared to gamma-glutamyltranspeptidase, and was more consistently expressed at high levels in tumours, suggesting that it might be a more reliable way of identifying those cells involved in the transformation process.

Highly specific characterization of tyrosine phosphoproteins in tumor cells based on monoclonal antibodies defined by conjugated phosphotyramine.

Phosphotyrosine proteins of four different tumor cell lines were characterized by monoclonal antibodies exhibiting high affinity binding to phosphotyrosine. For the preparation of the antibody-producing mouse hybridoma cell lines we used a novel kind of immunizing antigen with phosphotyramine conjugated directly to carboxylic groups of carrier proteins. Screening for high affinity binding antibodies was based on their selective reactivity in immunoprecipitation, affinity chromatography and immunofluorescence. By means of affinity chromatography we established a one-step purification of phosphotyrosine proteins yielding substantial quantities of highly pure 170kDa EGF receptor from A431 tumor cells, 210kDa bcr-abl gene product from K562 tumor cells and 120 kDa transforming protein of the Abelson murine leukemia virus from TK tumor cells. Cross-reactivity with phosphoproteins containing no phosphotyrosine was not observed.

A study of human small-cell lung carcinoma (hSCLC) cell lines with different sensitivities to detect relevant mechanisms of cisplatin (CDDP) resistance.

The cisplatin(CDDP)-resistant cell line GLC4-CDDP shows a variety of differences from the parent line GLC4. The aim of this study was to determine which of the observed changes correlated with the degree of resistance and was therefore relevant to the phenomenon of CDDP resistance. For these experiments we used cells of the sensitive hSCLC cell line GLC4 and the in vitro-acquired CDDP-resistant sublines GLC4-CDDP3 and GLC4-CDDP11, with a resistance factor (RF) of 3 and 11 respectively for CDDP and of 1.8 and 7.4 respectively for carboplatin. Carboplatin was used, in addition to CDDP in seeking relevant mechanisms. No consistency was found between the RF and the growth pattern or antigen expression, cellular volume, doubling time, cellular or nuclear platinum (Pt) content or the level of Pt-non-histone chromatin protein (NHCP) binding. A correlation was found between the RF and the level of glutathione (GSH), and a trend was found for the level of Pt-DNA binding, Pt-GG adduct content and the amount of interstrand cross-links (ISC). These changes might therefore be relevant for the development of resistance. These findings are compatible with a GSH-induced reduction of the amount of reactive Pt in the resistant cell, resulting in a lower net platination and toxic Pt-DNA adduct formation.

Increased fucosylation of high-molecular-weight glycoproteins accompanies retinoic-acid-induced differentiation of F-9 embryonal carcinoma cells.

Retinoic acid (RA) treatment of F-9 embryonal carcinoma cells resulted in cell flattening and increased production of laminin B1 chain, both indicating differentiation to endoderm-like cells. In addition, RA caused a time- and dose-dependent decrease in growth rate in monolayer culture and a dose-dependent decrease in the ability of the cells to form colonies in soft agarose. Differentiation was accompanied by an increase in the fucosylation of specific high-molecular-weight cellular and cell-surface glycoproteins. The fucosylation of glycoproteins of Mr 175,000 (gp175), 250,000 (gp250), and 400,000 (gp400) increased as early as 24 hr after the addition of 5 x 10(-6) M RA to the culture medium. These changes preceded both growth inhibition and the induction of laminin B1 expression, which were detected 48 to 72 hr after addition of RA. The increased fucosylation of these glycoproteins showed a distinct dose-response relationship. Both gp175 and gp250 showed the greatest increase in fucosylation at 10(-5) M, which was also the dose at which RA induced laminin maximally, while the fucosylation of gp400 was greatest at 10(-8) M RA and declined at higher concentrations. The overall synthesis of large fucosylated glycopeptides decreased in RA-treated cells, in spite of the increases in the fucosylation of specific cellular glycoproteins. RA-induced differentiation of F-9 cells was also accompanied by a time- and dose-dependent increase in fucosyltransferase activity. Although the functions of these glycoproteins are not currently known, the early increase in their fucosylation can be considered as a marker of differentiation in this system.

Glycosphingolipid expression on murine L1-fibrosarcoma cells: analysis of clonal in vivo and in vitro selected sublines with different lung colonisation potential.

The patterns of acidic and neutral glycosphingolipids (GSLs) were examined in a syngeneic tumour system in Balb/c mice consisting of closely related cell lines with different colonisation potentials directed to the murine lungs (in vivo selected highly metastatic sublines of L1-fibrosarcoma cells and their WGA-resistant mutants with low metastatic potential). GSLs were analysed by high-performance thin-layer chromatography and structurally identified by fast atom bombardment mass spectrometry combined with compositional analyses and exo-glycosidase digestion. The results suggest that highly metastatic sublines L1-LM and L1-LM12 derived by in vivo selection from mouse fibrosarcoma cells (cell line L1) exhibit a drastic increase of polar ganglioside expression and a restriction to globo-series GSLs. Contrasting with this the low metastatic mutant cells (L1-LM13WGA) express a reduced portion of acidic GSLs and exhibit a shift to less polar ganglioside components. Total cellular and plasma membrane-integrated GSLs were demonstrated to exhibit largely identical patterns. Concomitant with a significant decrease in LacCer expression a substantial reduction of GM2 and a complete lack of GM3 expression can be assigned to the highly metastatic sublines of L1-cells. On the other hand, the more polar gangliosides GM1a and, to an even greater extent, GD1a (exceeding 70% of total gangliosides) accumulate on L1-LM and their clonal sublines. The shift to acidic GSLs of higher polarity is less pronounced on the low metastatic WGA-resistant mutant cells (L1-LM13WGA) showing a preponderance of GM1a. The portion of GD1a within the fractions of acidic GSLs does not correspond to the cellular activities of CMP-NeuAc/GM1 (alpha 2-3) sialyltransferase measured for high and low metastatic cell variants. Total sialic acid content of the various cell lines differs, but is not associated with the metastatic potential. Gangliosides on L1-cells exhibit a significant substitution of N-glycolyl for N-acetylneuraminic acid (13%) compared to their metastatic sublines and to mutant cells (less than 1%). A conversion of surface exposed GD1a to GM1a on membranes of metastatic cells by in situ treatment with Vibrio cholerae sialidase is associated with a significant reduction of tumour cell colonisation directed to the murine lungs.

TSH subunit gene promoters from a murine alpha-subunit producing tumor function normally.

The murine thyrotropic MGH101A tumor is characterized by absent thyrotropin (TSH) beta gene expression and altered thyroid hormone (T3) regulation of the alpha-subunit. Comparison of the promoter structures of both alpha and TSH beta subunit genes from MGH101A with the promoter in expressing TtT-97 thyrotropes revealed no detectable differences. Transfection of the TSH beta promoter from MGH101A linked to luciferase showed minimal expression in primary or cloned MGH101A cells, or L-cells. However, a 6- to 10-fold increase in expression was exhibited in transfected thyrotropes. For the alpha gene, promoter activity was highest in thyrotropes and in cloned MGH101A cells, 5-fold lower in MGH101A tumors, and 10-fold lower in L-cells. Both promoters were not substantially affected by T3 treatment in MGH101A cells. In thyrotropes, promoter activity was inhibited 62.5% and 57.7% by 10 nM T3 treatment for the TSH beta and alpha genes, respectively. DNase I protection showed that factors from TtT-97 but not from MGH101A cells interacted with regions in the TSH beta promoter, while nuclear extracts from each tumor demonstrated at least one protein-DNA interaction with the alpha-subunit promoter. These studies suggest that the molecular defects in the MGH101A tumor are related to the absence of trans-acting factors and are not a result of altered primary gene structure.

Coding region structure of interleukin-8 gene of human lung giant cell carcinoma LU65C cells that produce LUCT/interleukin-8: homogeneity in interleukin-8 genes.

A 1.9-kb fragment containing an interleukin-8 (IL-8) coding region was amplified by the polymerase chain reaction (PCR) from the genomic DNA of human lung giant cell carcinoma LU65C cells that produce LUCT/IL-8 with N-terminal sequence of AVLPR. The coding region was found to consist of 4 exons and 3 introns as identical as that of the gene of MDNCF/IL-8 lacking N-terminal AVLPR. PCR using genomic DNAs from human polymorphonuclear leukocytes and mononuclear cells also provided the same 1.9-kb fragment as that from LU65C genomic DNA. Thus, it seems likely that human cells possess IL-8 genes with the homogeneous coding region so that they may first produce the same mature protein with N-terminal AVLPR (= LUCT) which was then truncated.

Estradiol stimulation and inhibition of cell growth in new estrogen-sensitive cell lines and tumors established from the MtTF4 tumor.

Cell lines were established from the MtTF4 tumor, growth of which is inhibited by estradiol, in order to determine whether the effect observed in vivo was due to a direct action on tumor cells. Two different cell lines were obtained according to the medium in which tumor cells were dispersed and cultured. The F4P cells were obtained when the culture medium contained charcoal-treated fetal calf serum. The growth rate of these cells was slowed down by 17 beta-estradiol in animals and also in culture during the early passages. Thereafter, they became insensitive to 17 beta-estradiol in culture but remained negatively controlled in vivo. These cells, whatever their sensitivity to 17 beta-estradiol, secrete prolactin and carry functional D2 dopamine binding sites. The F4Z cells were established in medium containing fetal calf serum not treated with charcoal. The growth rate of these cells was stimulated by 17 beta-estradiol in animals but was 17 beta-estradiol insensitive in culture up to subculture 26. At this time, the growth rate of the subline also became stimulated by 17 beta-estradiol in culture, and this phenotype was still found at passage 108 (50% effective dose, 5 to 10 pmol; maximum stimulation, 180 to 300% of control). These cells neither secrete measurable amounts of prolactin nor have dopamine binding sites. Thus, according to the medium in which cells were dispersed and cultured, two different cell strains were derived from a tumor in which growth is inhibited by 17 beta-estradiol. The point of interest is that the growth rate of one strain was inhibited by 17 beta-estradiol, while the other was stimulated. Convergent data suggest that MtTF4 tumor was heterogeneous and that selection had occurred during the dispersion or the culture of cells. Since the growth of one of these cell lines was slowed down transiently in culture we conclude that the inhibition of tumor growth could be due to a direct action of 17 beta-estradiol on tumor cells. However, the dissociation between the response to 17 beta-estradiol in culture and in the animal observed at some time of cell evolution suggests that environment affects the sensitivity of cells to 17 beta-estradiol.

Beta 2-microglobulin expression in human embryonal neuroblastoma reflects its developmental regulation.

Class I major histocompatibility complex (MHC) antigen expression in neuroblastoma may play a role in the oncogenicity of this embryonal tumor of childhood. Since N-myc amplification in neuroblastoma tumors is associated with rapid tumor progression (33) and N-myc decreases Class I MHC antigen expression in rat neuroblastoma cells (21), we quantitated levels of N-myc mRNA and Class I MHC cell surface antigens in a panel of 24 human neuroblastoma cell lines. We found that N-myc expression is not invariably associated with low levels of beta 2-microglobulin (B2M) and Class I MHC antigen expression. As we considered that Class I MHC antigens may be regulated in association with the differentiation stage of the neuroblastoma tumor, we examined the expression of B2M during development of the human adrenal medulla, the tissue of origin of most neuroblastomas. We found that B2M is a marker of differentiated adrenal medullary cells, expressed late during the third trimester of development. Moreover, using morphological and immunological criteria, we found that B2M is expressed in differentiated tumor cells. These data suggest that the expression of B2M in neuroblastoma is associated with the stage of differentiation of the tumor cell and not N-myc expression. Furthermore, these findings suggest that neuroblastomas may correspond to the arrested differentiation of adrenal neuroblasts at different stages of development.

Expression of a cytomegalovirus-human growth hormone-releasing hormone precursor fusion gene in transfected GH3 cells.

Pituitary GH3 cells were transfected with a human growth hormone-releasing hormone (hGRH) precursor minigene fused to the promoter region of either a cytomegalic immediate early gene (pCMV) or the mouse metallothionein-1 gene (mMT) to examine the molecular heterogeneity of the translation products. Expression of the hGRH message occurred following transfection of the cells with each fusion gene. Extracts of pCMV-hGRH-transfected GH3 cells as well as the culture medium contained detectable levels of immunoreactive (ir)-hGRH peptides. Analysis of molecular heterogeneity by reverse-phase high performance liquid chromatography and radioimmunoassay indicated that both mature forms of hGRH (hGRH(1-44)-NH2 and hGRH(1-40)-OH) were synthesized in the cells, although hGRH(1-44)-NH2 was the primary form secreted into the medium. A high molecular weight form of ir-hGRH, believed to represent the hGRH precursor (or a partially processed form of the precursor) was detected in cells and, in smaller amounts, in the medium. Several ir-hGRH peptides, presumed cleavage products of the mature forms of hGRH, were also found. The efficiency of processing of the hGRH precursor and metabolism of the mature hormonal forms in transfected cells grown in the presence of four different peptidase inhibitors varied with the inhibitor present. Transfected GH3 cells, therefore, possess all of the necessary enzymes for and are capable of processing the hGRH precursor to mature GRH and provide a model to study hGRH biosynthesis.