CTGF, intestinal stellate cells and carcinoid fibrogenesis.

AIM: To investigate the role of small intestinal carcinoid tumor-derived fibrotic mediators, TGFbeta1 and CTGF, in the mediation of fibrosis via activation of an "intestinal" stellate cell. METHODS: GI carcinoid tumors were collected for Q RT-PCR analysis of CTGF and TGFbeta1. Markers of stellate cell desmoplasia were identified in peritoneal fibrosis by immunohistochemistry and stellate cells cultured from fresh resected fibrotic tissue. CTGF and TGFbeta1 were evaluated using quantitative tissue array profiling (AQUA analysis) in a GI carcinoid tissue microarray (TMA) with immunostaining and correlated with clinical and histologically documented fibrosis. Serum CTGF was analyzed using a sandwich ELISA assay. RESULTS: Message levels of both CTGF and TGFbeta1 in SI carcinoid tumors were significantly increased (> 2-fold, P < 0.05) versus normal mucosa and gastric (non-fibrotic) carcinoids. Activated stellate cells and markers of stellate cell-mediated fibrosis (vimentin, desmin) were identified in histological fibrosis. An intestinal stellate cell was immunocytochemically and biochemically characterized and its TGFbeta1 (10-7M) initiated CTGF transcription response (> 3-fold, P < 0.05) demonstrated. In SI carcinoid tumor patients with documented fibrosis, TMA analysis demonstrated higher CTGF immunostaining (AQUA Score: 92 +/- 8; P < 0.05), as well as elevated TGFbeta1 (90.6 +/- 4.4, P < 0.05). Plasma CTGF (normal 12.5 +/- 2.6 ng/mL) was increased in SI carcinoid tumor patients (31 +/- 10 ng/mL, P < 0.05) compared to non-fibrotic GI carcinoids (< 15 ng/mL). CONCLUSION: SI carcinoid tumor fibrosis is a CTGF/TGFbeta1-mediated stellate cell-driven fibrotic response. The delineation of the biology of fibrosis will facilitate diagnosis and enable development of agents to obviate its local and systemic complications.

Role of CCN2/CTGF in the proliferation of Mastomys enterochromaffin-like cells and gastric carcinoid development.

Mastomys enterochromaffin-like (ECL) cell proliferation is initially gastrin driven, but once neoplasia develops, cells become gastrin autonomous. We hypothesized that CCN2 (CTGF), a mitogenic growth factor, may regulate ECL cell proliferation. A Mastomys GeneChip database was examined (dCHIP) to identify CCN2 expression levels. CCN2 in normal and tumor ECL cell preparations obtained using FACS (100 nM acridine orange) was examined by real-time PCR. CCN2 protein was identified in mucosal and ECL cell preparations by immunohistochemistry. Short-term cultured cells were stimulated with either CCN2 or CCN2 + EGF, and proliferation was measured (MTT assay). The ERK1/2 inhibitor PD-98059 (0.1-100 microM) was assessed in terms of CCN2 (1 ng/ml)-mediated proliferation and ERK1/2 phosphorylation. CCN2 transcript and protein was then examined in clinical gastric carcinoids. The ccn2 transcript was upregulated in tumor samples compared with the normal mucosa (+2.36-fold, P < 0.01). PCR demonstrated that ccn2 was not expressed in FACS-prepared (>98% pure) normal ECL cells but was elevated in tumor ECL cell fractions (41.3 +/- 10.7-fold). Immunostaining of the Mastomys gastric mucosa and FACS preparations confirmed that CCN2 protein was present in ECL tumors but not in normal ECL cells. Neither CCN2 nor CCN2 + EGF stimulated normal ECL cell proliferation. CCN2 stimulated tumor proliferation (EC50 approximately 0.01 ng/ml); EGF significantly augmented (P < 0.01) CCN2-induced tumor cell proliferation (EC50 = 20 pg/ml). PD-98059 inhibited CCN2-induced proliferation (-12 +/- 3%, P < 0.05) and ERK1/2 phosphorylation (-34 +/- 5%, P < 0.05) in tumor cells. In clinical samples, both CCN2 transcript and protein were elevated in gastrin-autonomous carcinoids (P < 0.02) compared with the normal mucosa. In conclusion, CCN2 may be a proliferative regulator of Mastomys ECL neoplastic proliferation once these cells become autonomous of gastrin regulation. Identification of CCN2 in gastric carcinoid tissue may be useful both as an indicator of ECL cell transformation and may define gastrin autonomy, a criteria of gastric carcinoid malignancy.

Global expression analysis of ECL cells in Mastomys natalensis gastric mucosa identifies alterations in the AP-1 pathway induced by gastrin-mediated transformation.

Enterochromaffin-like (ECL) cell hyperplasia and then irreversible neoplasia can be generated in the African rodent Mastomys natalensis using the H2 receptor blocker, loxtidine, for 8-16 wk. We used a GeneChip approach complemented by standard technologies to identify gene expression alterations in the gastric mucosa during gastrin-mediated ECL cell transformation. Gastric mucosa (mucosal scrapping) and ECL cell-enriched fractions were obtained from untreated Mastomys (controls) and from animals treated with loxtidine for 8 wk (hyperplasia). Tumor ECL cells were obtained by hand-dissection of gastric ECL cell nodules from animals treated with loxtidine for >16 wk and from a spontaneously developed ECL cell tumor. RNA was isolated, examined on rat U34A GeneChips, and comparison analysis was performed to identify altered gene expression. Alterations in gene expressions were examined further by immunohistochemistry, quantitative RT-PCR (Q-RT-PCR), sequencing and Western blot. GeneSpring analysis demonstrated alterations in few genes (<20) in hyperplastic and tumor mucosa. The histamine H1 receptor was consistently increased in proliferating mucosa. This gene change was confirmed by Q-RT-PCR. Other genes showing alterations included neural-(chromogranin A and somatostatin), cell-cycle-, and AP-1-associated genes. Immunostaining confirmed alterations in neural markers. Cluster analysis of ECL cell-enriched samples demonstrated that c-fos and junD were differently regulated. Q-RT-PCR and Western blot in prospectively collected gastric mucosal samples confirmed the differential expression of Fos and Jun. The negative regulators of AP-1, JunD, and Menin were decreased in tumor mucosa. A missense of unknown function was noted in the menin gene. Hypergastrinemia in an animal model of gastric carcinoids differentially altered the histamine type 1 receptor and gene expression and protein composition of AP-1. These results suggest that expression of this receptor and an altered composition of AP-1 with a loss of inhibition play a role in ECL cell transformation.

Fibrinogen Baltimore I: polymerization defect associated with a gamma 292Gly----Val (GGC----GTC) mutation.

Fibrinogen Baltimore I is one of the very first congenital abnormal fibrinogens reported over several decades ago; however, the molecular defect of this dysfibrinogen has eluded identification. In fact, several reports misidentified the functional defect of Baltimore I, which has impaired fibrin monomer polymerization. Reversed-phase high-performance liquid chromatography analysis of lysyl endopeptidase digest of the purified Baltimore I gamma-chain showed an abnormal peptide not found in the co-existing normal gamma-chain of this heterozygote. Amino acid sequencing of this peptide indicated that gamma-chain Gly292 is replaced by valine. This observation was confirmed, and the genetic defect was determined by direct nucleotide sequencing of a polymerase chain reaction product containing codon gamma 292, which is mutated: GGC----GTC. The molecular defect of Fibrinogen Baltimore I lies in a region of the gamma-chain required for fibrin polymerization, suggesting that the integrity of gamma Gly292 is critical for fibrin assembly.

Quantitation of the ras gene product in leukemic blast cells using enzymatic staining.

Few studies have focused on the significance of ras protein levels in human malignancy, in part because of the inherent difficulty in quantitation of the ras gene product. We have developed a method for the enzymatic determination of the ras gene product and have used this method for the quantitation of ras gene product levels in 19 patients with acute leukemia. This technique provides a practical means to assess p21 expression in leukemic cells ex vivo while avoiding the use of radioactive reagents. In addition, the mobility of the ras species of interest is determined. This assay should be easily modified for the use of other antibodies such as those reported to be specific for various ras species (i.e., H-, K- and N-ras), for specific ras mutations or for other nonras proteins. Because of the use of electrophoresis prior to quantitation of protein, the antibody used does not need to possess high specificity for the protein of interest.

Activation of c-Ki-ras in human gastrointestinal dysplasias determined by direct sequencing of polymerase chain reaction products.

Activation of c-Ki-ras by point mutation within exon 1 was studied in 33 specimens of dysplastic gastrointestinal lesions or of cancers presumed to arise from dysplasia. Samples were obtained from patients with underlying ulcerative colitis or Barrett's esophagus, two diseases associated with dysplasia and increased rates of colonic or esophageal adenocarcinoma, respectively. Genomic DNA was amplified using primers bounding this exon in the polymerase chain reaction. Polymerase chain reaction products were analyzed by direct dideoxy sequencing. Three point mutations in codon 13 of c-Ki-ras were found, all in colonic specimens (two high-grade dysplasias and one adenocarcinoma arising in ulcerative colitis). No point mutations were observed in the second exon of c-Ki-ras or in and around codons 12, 13, and 61 of c-N-ras and C-Ha-ras in a partial sampling of the specimens. These data indicate that ras family protooncogene activation is an uncommon event at this level of malignant progression in these disease states. Carcinogenesis in ulcerative colitis and Barrett's esophagus may proceed via different pathways than in sporadic colon cancer, perhaps involving loss or inactivation of suppressor genes.

RAS gene activation in acute myelogenous leukemia: analysis by in vitro amplification and DNA base sequence determination.

RAS protooncogene activation has been repeatedly demonstrated in neoplastic cell DNA from patients with AML. Despite the convincing demonstration that activating RAS gene point mutations are critical in model systems, their precise prevalence and importance in human cancers such as AML remain speculative. The technology for identifying RAS mutations has changed considerably in recent years. We examined a prospective cohort of 43 acute myeloid leukemia (AML) patients admitted to the University of Maryland Cancer Center for first and second exon mutations of NRAS and KRAS using PCR and DNA sequence analysis. Six (14%) 1st exon NRAS mutations were identified. No clinical or biologic parameter has yet been observed to segregate with RAS activation, although a larger study may be needed to demonstrate this.

Ras gene product expression in blood and marrow smears of patients with acute leukemia: importance of fixation.

Activation of ras protooncogenes by any of several possible mutations in codons 12, 13 or 61 has been demonstrated in a variety of human malignancies, including acute non-lymphoblastic leukemia (ANLL). In situ staining for the ras gene product, p21, has been demonstrated in carcinomas of several sites. High levels of p21 expression have been associated with histologic anaplasia in prostate cancer and regional lymph node metastasis in breast cancer. We examined 16 marrow aspirates and blood smears from patients with acute leukemia, predominantly ANLL, and eight controls. Marrow aspirates or blood were smeared on glass slides and fixed immediately in 10% buffered formalin. p21 was examined with avidin-biotin linked immunoperoxidase visualization. Particular attention must be paid to antibody selection and fixation protocol to demonstrate p21, owing to its rapid degradation ex vivo. Three of 16 patients exhibited occasional high p21 expression primarily in leukemic blasts, but in no case were more than 10% of blast cells positive. Normal reticuloendothelial and myeloid cells occasionally exhibited mild to moderately heavy staining, but megakaryocytes, erythroid precursors, lymphocytes and plasma cells were consistently negative. Most patients, 5 normal volunteers and 3 patients with non-malignant disease, exhibited no reactivity, or only a faint blush. These data suggest that while point mutation and concomitant activation of c-N-ras occurs regularly in ANLL, high levels of ras p21 expression are rarely found with this technique.

Sequencing products of the polymerase chain reaction directly, without purification.

An improvement over current protocols for sequencing products of the polymerase chain reaction is described. This method allows sequencing products of the reaction without performing costly, time-consuming purification steps which often result in unacceptable loss of product. Conservation of small amounts of polymerase chain reaction products which can be obtained from limited DNA sources, such as tissue biopsies, is achieved. Clarity of autoradiograms obtained utilizing this adaptation is comparable to that obtained with the original method. In addition to streamlining the amplification-sequencing procedure, this procedure can potentially be subjected to total automation.

Hypereosinophilic syndrome with evolution to myeloproliferative disorder: temporal relationship to loss of Y chromosome and c-N-ras activation.

A case of hypereosinophilic syndrome is presented in which the patient was serially observed for 4 years. Transformation to a disorder resembling chronic myeloid leukemic (CML) occurred 36 months after diagnosis; at 42 months, blastic transformation and marrow failure ensued, leading to death. Marrow examination for histopathologic, cytogenetic, and molecular biologic analyses were performed during the eosinophilic, myeloproliferative, and blastic stages. These demonstrated ras activation by virtue of a codon 12 G to C transversion mutation, predicting for substitution of glycine by alanine; in addition, we observed Y chromosome loss late in the natural history of this illness, suggesting that these genetic lesions can play a role in the profound loss of myeloid differentiation characteristic of the accelerated phase commonly observed in myeloproliferative syndromes.

Infrequent ras activation in chronic myelogenous leukemia (CML): activating 61st codon mutation in the CML-derived cell line, IM-9.

The proto-oncogene c-N-ras frequently bears point mutations in ANLL cell DNA which endow it with the capacity to transform NIH/3T3 cells in vitro. Chronic myelogenous leukemia (CML) is a neoplasm highly related to ANLL since it involves the same hematopoietic progenitor cells and ultimately transforms to a neoplasm virtually indistinguishable from acute nonlymphoblastic leukemia (ANLL). Thus, we and others have examined ras genes in CML. This report confirms that ras gene activation is a very infrequent event in CML. However, a lymphoblastic cell line derived from a patient with CML did exhibit a novel second exon 61st codon activating mutation of c-N-ras.

c-myc amplification coexistent with activating N-ras point mutation in the biphenotypic leukemic cell line RED-3.

While activation of the protooncogene c-N-ras is observed regularly in acute myelogenous leukemia, amplification of c-myc in AML cells or derived lines is uncommon. In particular, concurrent ras/myc activation, which has been shown to be critical in several elegant models of malignancy, has been demonstrated in a very small number of human tumors or derivative cell lines. A cell line, RED-3, is described which was derived from cells of a patient with aggressive acute leukemia which exhibits many markers of lineage infidelity. DNA from this cell line contains an activating point mutation of c-N-ras as well as 20-30-fold amplification of c-myc. After HL-60, this is the second example of ras/myc activation in AML derived cells and demonstrates that this lesion is not unique to HL-60. Rather, it may be important in leukemogenesis in a small proportion of AML patients.

Purification and characterization of human lysosomal membrane glycoproteins.

Two human cell lysosomal membrane glycoproteins of approximately 120 kDa, hLAMP-1 and hLAMP-2, were identified by use of monoclonal antibodies prepared against U937 myelomonocytic leukemia cells or blood mononuclear cells. The two glycoproteins were purified by antibody affinity chromatography and each was found to be a major constituent of human spleen cells, representing approximately 0.05% of the total detergent-extractable protein. Both molecules were highly glycosylated, being synthesized as polypeptides of 40 to 45 kDa and cotranslationally modified by the addition of Asn-linked oligosaccharides. NH2-terminal sequence analysis indicated that each was approximately 50% identical to the corresponding mLAMP-1 or mLAMP-2 of mouse cells. Electron microscopic studies of human blood monocytes, HL-60, and U937 cells demonstrated that the principal location of these glycoproteins was intracellular, in vacuoles and lysosomal structures but not in the peroxidase-positive granules of monocytes. Transport of the proteins between organelles was evidenced by their marked accumulation in the membranes of phagolysosomes. A fraction of each glycoprotein was also detected on the plasma membrane of U937 and HL-60 cells but not on a variety of other tissue culture cells. This cell-surface expression may be differentiation related, since the proteins were not detected in the plasma membrane of normal blood monocytes and their expression on U937 and HL-60 cells was reduced when the cells were treated with differentiating agents. Cell-surface expression of both glycoproteins was markedly increased in blood monocytes but not in U937 cells after exposure to the lysosomotropic reagent methylamine HCl, indicating differences in LAMP-associated membrane flow in these cell types.