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H C Wu

Publications and source records attributed to H C Wu.

At least 19 recordsLinked to original sources
PubMed1995

Ceramide reverses brefeldin A (BFA) resistance in BFA-resistant cell lines.

We have found that C6 ceramide, a cell-permeable ceramide analog, partially restored the brefeldin A (BFA) sensitivity in a BFA-resistant mutant of Vero cells (BER-40) and in the naturally BFA-resistant Madin-Darby canine kidney (MDCK) cells. Incubation of BER-40 and MDCK cells with low concentrations of C6 ceramide resulted in (i) a pronounced increase in BFA cytotoxicity as measured by the inhibition of [3H]thymidine incorporation and the inhibition of colony formation by BFA, (ii) a significant protection by BFA against ricin cytotoxicity, and (iii) an inhibition of bulk protein secretion by BFA in BER-40 and MDCK cells. Related sphingolipids including sphingosine, sphingomyelin, and lactosylceramide and other unrelated lipid second messengers such as arachidonic acid and 1,2-diacylglycerol did not elicit the protection of BER-40 and MDCK cells against ricin cytotoxicity by BFA. C6 ceramide was the most effective among the ceramides with different acyl chain lengths. Interestingly, dihydro-C6 ceramide, which lacks the trans double bond in the sphingoid base, had no effect. On the other hand, C6 ceramide did not enhance BFA sensitivity in BFA-sensitive Vero cells. The LD50 of C6 ceramide were similar in Vero and BER-40 cells. Fluorescence microscopic studies revealed that C6 ceramide induced the redistribution of beta-COP from the Golgi membranes to a more dispersed localization in both BFA-sensitive and BFA-resistant cell lines, mimicking the effect of BFA. Suboptimal concentration of C6 ceramide also restored the effect of BFA on the beta-COP distribution in BER-40 and MDCK cells. These results indicate that C6 ceramide restores the BFA sensitivity in BFA-resistant BER-40 and MDCK cells.

Animals↗
PubMed1995

Ilimaquinone inhibits the cytotoxicities of ricin, diphtheria toxin, and other protein toxins in Vero cells.

Ilimaquinone (IQ), a metabolite from sea sponges, has been shown to cause the breakdown of Golgi membranes into small vesicular structure and to inhibit protein transport without eliciting the retrograde transport of the Golgi enzymes to the endoplasmic reticulum [P. A. Takizawa, J. K. Yucel, B. Viet, D. J. Faulkner, T. Deerinck, G. Soto, M. Ellismann, and V. Malhotra, Cell (1993) 73, 1079-1090]. We have found that incubation of Vero cells with IQ inhibited the cytotoxicity of ricin in a dose-dependent manner. The inhibition was reversed upon the removal of IQ. Neither binding and internalization of 125I-ricin nor the translocation of ricin to the cytosol was affected by IQ. However, IQ significantly inhibited the recycling and degradation of internalized 125I-ricin. Preincubation with IQ also prevented the enhancement of ricin cytotoxicity by NH4Cl or nigericin. The inhibition of ricin cytotoxicity by IQ was observed in the presence of cycloheximide, indicating that de novo protein synthesis is not required for IQ-mediated protection of Vero cells from ricin cytotoxicity. In contrast to perinuclear distribution of TRITC-labeled ricin in Vero cells, TRITC-ricin appeared in numerous small vesicles dispersed throughout the cytoplasm in IQ-treated Vero cells. Double labeling with C6-NBD-ceramide and TRITC-labeled ricin showed that these ricin-containing vesicles were distinct from the IQ-induced breakdown product of the Golgi membranes. Like brefeldin A (BFA), IQ inhibited the cytotoxicities of abrin, modeccin, Pseudomonas toxin, and Shiga-like toxin in Vero cells. Unlike BFA, IQ also inhibited the cytotoxicity of diphtheria toxin (DT). Inhibition of DT cytotoxicity was the consequence of a decreased specific binding of the toxin in the IQ-treated cells.

Animals↗
PubMed1995

Protective effect of cell-permeable ceramide analogs against modeccin, ricin, Pseudomonas toxin, and diphtheria toxin.

We investigated the effects of various ceramide (Cer) analogs and related sphingolipids on the cytotoxicities of modeccin, ricin, Pseudomonas toxin, and diphtheria toxin in various cell lines. The most pronounced protective effect by C6Cer, a short-chain cell-permeable Cer analog, was observed in modeccin cytotoxicity in Vero, BER-40, and MDCK cells, whereas the cytotoxicity of diphtheria toxin was not affected by any of the ceramide analogs tested. C6Cer did not affect the binding and internalization of ricin and modeccin in Vero and BER-40 cells. C2Cer and C8Cer also protected against modeccin cytotoxicity, albeit less effectively than C6Cer. However, related sphingolipids including sphingosine, sphingomyelin, lactosylceramide, C18Cer (the naturally occurring ceramide), and dihydro C6Cer had no effect. A correlation was found between the ability of ceramides to inhibit bulk protein secretion and the inhibition of modeccin cytotoxicity by ceramides. Among Cer analogs tested, C6Cer, the most potent inhibitor of modeccin cytotoxicity, strongly inhibited bulk protein secretion in Vero, BER-40, and MDCK cells. PtK1 cells, which were not protected by ceramides against toxins, were resistant to ceramide-induced inhibition of bulk protein secretion. These results confirm that Cer may modulate the intracellular transport of proteins through the Golgi complex. Such Cer-sensitive processes may be involved in the intoxication of cells by plant and bacterial toxins, especially modeccin.

Animals↗
PubMed1995

Identification of cutC and cutF (nlpE) genes involved in copper tolerance in Escherichia coli.

It has been suggested previously that copper transport in Escherichia coli is mediated by the products of at least six genes, cutA, cutB, cutC, cutD, cutE, and cutF. A mutation in one or more of these genes results in an increased copper sensitivity (D. Rouch, J. Camakaris, and B. T. O. Lee, p. 469-477, in D. H. Hamer and D. R. Winge, ed., Metal Ion Homeostasis: Molecular Biology and Chemistry, 1989). Copper-sensitive cutC and cutF mutants were transformed with a genomic library of E. coli, and copper-tolerant transformants were selected. Two distinct clones were identified, each of which partially restores copper tolerance in both the cutC and cutF mutants of E. coli. Subcloning, physical mapping, and sequence analysis have revealed that the cutC gene is located at 42.15 min on the E. coli genome and encodes a cytoplasmic protein of 146 amino acids and that the cutF gene is located at 4.77 min on the E. coli genome and is allelic to the nlpE gene independently identified by Silhavy and coworkers (W. B. Snyder, L. J. B. Davis, P. N. Danese, C. L. Cosma, and T. J. Silhavy, J. Bacteriol. 177:4216-4223, 1995). Results from the genetic mapping of the copper-sensitive mutations in the cutF mutant and sequencing of the cutC and cutF (nlpE) alleles from both cutC and cutF mutants indicate that both the cutC and cutF mutants are in fact double mutants altered in these two genes, and mutations in both the genes appear to be required for the copper-sensitive phenotype in each mutant.

Alleles↗
PubMed1995

Structure-function relationship of bacterial prolipoprotein diacylglyceryl transferase: functionally significant conserved regions.

The structure-function relationship of bacterial prolipoprotein diacylgyceryl transferase (LGT) Has been investigated by a comparison of the primary structures of this enzyme in phylogenetically distant bacterial species, analysis of the sequences of mutant enzymes, and specific chemical modification of the Escherichia coli enzyme. A clone containing the gene for LGT, lgt, of the gram-positive species Staphylococcus aureus was isolated by complementation of the temperature-sensitive lgt mutant of E. coli (strain SK634) defective in LGT activity. In vivo and in vitro assays for prolipoprotein diacylglyceryl modification activity indicated that the complementing clone restored the prolipoprotein modification activity in the mutant strain. Sequence determination of the insert DNA revealed an open reading frame of 837 bp encoding a protein of 279 amino acids with a calculated molecular mass of 31.6 kDa. S. aureus LGT showed 24% identity and 47% similarity with E. coli, Salmonella typhimurium, and Haemophilus influenzae LGT.S. aureus LGT, while 12 amino acids shorter than the E. coli enzyme, had a hydropathic profile and a predicted pI (10.4) similar to those of the E. coli enzyme. Multiple sequence alignment among E. coli, S. typhimurium, H. influenzae, and S. aureus LGT proteins revealed regions of highly conserved amino acid sequences throughout the molecule. Three independent lgt mutant alleles from E. coli SK634, SK635, and SK636 and one lgt allele from S. typhimurium SE5221, all defective in LGT activity at the nonpermissive temperature, were cloned by PCR and sequenced. The mutant alleles were found to contain a single base alteration resulting in the substitution of a conserved amino acid. The longest set of identical amino acids without any gap was H-103-GGLIG-108 in LGT from these four microorganisms. In E. coli lgt mutant SK634, Gly-104 in this region was mutated to Ser, and the mutant organism was temperature sensitive in growth and exhibited low LGT activity in vitro. Diethylpyrocarbonate inactivated the E. coli LGT with a second-order rate constant of 18.6 M-1S-1, and the inactivation of LGT activity was reversed by hydroxylamine at pH 7. The inactivation kinetics were consistent with the modification of a single residue, His or Tyr, essential for LGT activity.

Amino Acid Sequence↗
PubMed1995

The umpA gene of Escherichia coli encodes phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (lgt) and regulates thymidylate synthase levels through translational coupling.

Using a combination of biochemical, physical, and genetic techniques, we have shown that the umpA gene of Escherichia coli is allelic with the lgt (phosphatidylglycerol:prolipoprotein diacylglyceryl transferase) of Salmonella typhimurium. These genes are essential for the viability of the respective organism and exhibit 92.8% sequence identity at the amino acid level. In E. coli, lgt and thyA (thymidylate synthase) form an operon. Thymidylate synthase levels are regulated by transcription from the lgt promoter and by translational coupling.

Amino Acid Sequence↗
PubMed1995

Hepatopulmonary syndrome in liver cirrhosis: report of a case.

A 52-year-old woman suffered from exertional dyspnea, 5 years after she was diagnosed with cirrhosis of the liver. Hypoxemia was suspected because of persistent exertional dyspnea and was confirmed by contiguous arterial blood gas tests. After excluding other cardiopulmonary factors, a series of studies including lung perfusion scan, echocardiography and cardiopulmonary cineangiography revealed the cause of hypoxemia to be the patient's liver disease. Hepatopulmonary syndrome was diagnosed. Despite supplemental oxygen therapy and oral garlic powder for 6 months, the patient still had exertional dyspnea and platypnea, and arterial blood gas results did not improve. We report this case to call attention to arterial oxygen desaturation in cases of cirrhosis of the liver, and to emphasize that deteriorating oxygenation in patients with chronic liver disease may be an indication for liver transplantation, as such functional microvascular abnormalities could be reversed by liver transplantation.

Female↗
PubMed1994

Relationship of protein structure of isoleucyl-tRNA synthetase with pseudomonic acid resistance of Escherichia coli. A proposed mode of action of pseudomonic acid as an inhibitor of isoleucyl-tRNA synthetase.

To elucidate the mode of action of pseudomonic acid, we have compared the deduced amino acid sequences of isoleucyl-tRNA synthetases (ILeRS) from wild-type Escherichia coli strain MC4100, a pseudomonic acid-resistant mutant (strain PS102) of MC4100, and a pseudomonic acid-producing strain, Pseudomonas fluorescens. Compared with the wild-type enzyme, the deduced amino acid sequence of E. coli mutant ileS gene in strain PS102 shows a single amino acid substitution of leucine for phenylalanine at residue 594 of the IleRS. This mutational alteration in IleRS of an E. coli pseudomonic acid-resistant mutant resides in a region of the enzyme in close proximity to one of the consensus sequences of class I aminoacyl-tRNA synthetases, the KMSKS sequence between residues 602 and 606 of the E. coli IleRS. DNA sequence of the cloned ileS gene predicts that the P. fluorescens IleRS consists of 943 amino acids with 54% identity with the E. coli IleRS. The P. fluorescens ileS gene and the wild type and PS102 alleles of E. coli ileS were cloned into an expression vector, pEXPCR, and the sensitivities of E. coli DH5 alpha cells harboring each of these plasmids were compared. The cells harboring the P. fluorescens ileS were found to be most resistant to pseudomonic acid, while the transformants expressing the PS102 IleRS were more resistant than those containing the wild-type E. coli IleRS. IleRS purified from the wild-type E. coli was specifically cleaved by trypsin between Lys605 and Ser606 in the region of K602MSKS606. The protection of the IleRS from the trypsin digestion was found with pseudomonic acid or ATP, but not with isoleucine or tRNA(1Ile). Based on these results, we propose that pseudomonic acid binds to IleRS in the vicinity of the KMSKS sequence that is an ATP-binding subsite, and that pseudomonic acid is a bifunctional inhibitor with characteristics of both isoleucine and ATP, for example, an analog of isoleucyladenylate.

Adenosine Triphosphate↗
PubMed1994

Lipid modification of bacterial prolipoprotein. Transfer of diacylglyceryl moiety from phosphatidylglycerol.

The peptide, MKATKLVLGAVILGSTLLAGCSSN, corresponding to the N-terminal 24 amino acids of Braun's prolipoprotein, was used to study the lipid modification of prolipoprotein in Escherichia coli by measuring the rate of incorporation of either [2-3H]glycerol or [9,10-3H]palmitate from the corresponding labeled phosphatidylglycerol into the peptide. Using E. coli strains containing varying levels of prolipoprotein diacylglyceryl modification activities due to mutations in or overexpression of the gene involved in diacylglyceryl modification (lgt), we have shown that the activities based on the peptide assay correlated well with the prolipoprotein-based assay. Further, we have followed the fate of the lipid substrate, phosphatidylglycerol, during the modification reaction and found that lipid modification of prolipoprotein involves the transfer of diacylglyceryl moiety from phosphatidylglycerol to the sulfhydryl group of the cysteine residue with the concomitant formation of sn-glycerol 1-phosphate. This mechanism is contrary to the previously proposed two-step mechanism of an initial glyceryl transferase followed by O-acyl transfer (Chattopadhyay, P.K., and Wu, H.C. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5318-5322). Accordingly, the enzyme that catalyzes this activity has been named phosphatidylglycerol-prolipoprotein diacylglyceryl transferase. The revised pathway for the lipoprotein biogenesis in bacteria consists of three successive reactions catalyzed by prolipoprotein diacylglyceryl transferase, signal peptidase II, and apolipoprotein N-acyltransferase.

Amino Acid Sequence↗
PubMed1994

Derivation of androgen-independent human LNCaP prostatic cancer cell sublines: role of bone stromal cells.

A model of human prostate cancer was established to study cellular interaction between prostate cancer and bone stroma in vivo. In this model, subcutaneous co-injection of 2 non-tumorigenic human cell lines--LNCaP, a prostate cancer cell line, and MS, a bone stromal cell-line--into intact adult male mice resulted in formation of carcinomas that secreted prostate-specific antigen (PSA), a clinically useful human serum prostate cancer marker. In castrated hosts, upon cellular interaction with bone fibroblasts, we observed the progression of these tumors from an androgen-dependent (AD) to an androgen-independent state (AI). We derived 4 LNCaP cell sublines from the chimeric LNCaP/MS tumors: the M subline from intact hosts and the C4, C4-2 and C5 sublines from castrated hosts. The LNCaP sublines had chromosomal markers similar to those of the parental LNCaP cells and distinctly different from those of the MS bone stromal cell line. Although the parental and derived cell lines expressed similar steady-state levels of ornithine decarboxylase transcript, the sublines expressed 5- to 10-fold higher basal steady-state levels of PSA transcript than did the parental LNCaP cell line. The LNCaP sublines formed 13- to 26-fold more soft-agar colonies than the parental LNCaP cell line. The sublines became tumorigenic, yielding an incidence of tumors in intact athymic mice of 7-75%. The LNCaP sublines C4 and C5 (but not the parental and M cell line) formed tumors in castrated hosts when co-injected with bone fibroblasts. A second-generation LNCaP subline, C4-2, was derived from a chimeric tumor induced by co-inoculating castrated mouse with C4 cells and MS cells. We found that C4-2 subline was tumorigenic when inoculated into castrated hosts in the absence of inductive fibroblasts. Moreover, C4-2 was the only subline capable of forming soft-agar colonies when cultured in serum-free medium. In comparison with the parental LNCaP cells, the C4-2 subline expressed lower steady-state levels of androgen receptor (AR) protein and mRNA transcript and lost its androgen responsiveness in vitro. Our results suggest that certain genetic traits of prostate cancer cells may be selected or altered through an "adaptive" mechanism that involves cellular interaction with the bone stromal cells.

Animals↗
PubMed1994

Cytotoxic necrotizing factor type 2 produced by virulent Escherichia coli modifies the small GTP-binding proteins Rho involved in assembly of actin stress fibers.

Cytotoxic necrotizing factor type 2 (CNF2) produced by Escherichia coli strains isolated from intestinal and extraintestinal infections is a dermonecrotic toxin of 110 kDa. We cloned the CNF2 gene from a large plasmid carried by an Escherichia coli strain isolated from a lamb with septicemia. Hydropathy analysis of the deduced amino acid sequence revealed a largely hydrophilic protein with two potential hydrophobic transmembrane domains. The N-terminal half of CNF2 showed striking homology (27% identity and 80% conserved residues) to the N-terminal portion of Pasteurella multocida toxin. Methylamine protection experiments and immunofluorescence studies suggested that CNF2 enters the cytosol of the target cell through an acidic compartment and induces the reorganization of actin into stress fibers. Since the formation of stress fibers in eukaryotic cells involves Rho proteins, we radiolabeled these small GTP-binding proteins from CNF2-treated and control cells with a Rho-specific ADP-ribosyltransferase. The [32P]ADP-ribosylated Rho proteins from CNF2-treated cells migrated slightly more slowly in SDS/PAGE than did the labeled proteins from the control cells. This shift in mobility of Rho proteins in SDS/PAGE was also observed when CNF2 and the RhoA protein were coexpressed in E. coli. We propose that Rho proteins are the targets of CNF2 in mammalian cells.

Actin Cytoskeleton↗
PubMed1994

Effect of lovastatin on the cytotoxicity of ricin, modeccin, Pseudomonas toxin, and diphtheria toxin in brefeldin A-sensitive and -resistant cell lines.

Lovastatin, a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, inhibits the biosynthesis of cholesterol and the prenylation of proteins. In this study, we have found that lovastatin inhibited the cytotoxicities of modeccin, ricin, Pseudomonas toxin, and diphtheria toxin in Vero and a brefeldin A (BFA)-resistant mutant of Vero cells (BER-40) to different extents. Among these toxins tested, the cytotoxicity of modeccin was most strongly inhibited by lovastatin in a dose-dependent manner. The protective effect of lovastatin was completely reversed by the addition of mevalonic acid, while the addition of cholesterol had no effect on the cytotoxicity of modeccin in lovastatin-treated cells. These results suggest that prenylated proteins are involved in the intoxication process of modeccin. The addition of cycloheximide to the growth medium also reversed the protective effect of lovastatin, suggesting a requirement of de novo protein synthesis for the protection by lovastatin against toxins. In contrast to Vero and BER-40 cells, no significant effect of lovastatin was observed in naturally BFA-resistant cell lines, PtK1 and MDCK cells, even though similar morphological changes and disassembly of the actin microfilaments were induced by lovastatin in these cell lines as observed in Vero and BER-40 cells. Lovastatin did not affect the binding and internalization of ricin and modeccin in Vero and BER-40 cells. Our results suggest that prenylated cellular proteins are involved in intracellular trafficking or processing of protein toxins, especially modeccin. In PtK1 and MDCK cells, such intracellular vesicle trafficking of protein toxins may be regulated by lovastatin-resistant mechanisms.

ADP Ribose Transferases↗
PubMed1994

Bowel perforation--a fatal complication following renal transplantation: a report of two cases.

From May 1983 to January 1993, 162 patients received renal transplantation at Taichung Veterans General Hospital. Two patients developed bowel perforation after this surgery. One was proved at 21 days and another, at 13 days after renal transplantation. Both patients died as a direct result of the perforation. From a review of literature, the average incidence is 2.7%, and the average mortality rate is 56.5%. Because of this high mortality rate, a high clinical suspicion, prompt and adequate surgical intervention, reduction of immunosuppressive agents and effective antibiotic coverage should contribute to a decline in mortality.

Adult↗
PubMed1994

Association of heterotrimeric GTP binding regulatory protein (Go) with mitosis.

BACKGROUND: The heterotrimeric GTP-binding regulatory proteins (or G proteins) play an important role in regulation of membrane signal transduction. Previously, we have identified the beta-subunit of G proteins (G beta) to be closely associated with mitotic spindles. In the present study, we have conducted experiments to examine whether the G alpha subunit is also associated with mitotic spindles. EXPERIMENTAL DESIGN: We prepared polyclonal antibodies against synthetic oligopeptides from different G alpha subunits and used them to localize G alpha in a nasopharyngeal carcinoma cell line by both immunohistochemistry and immunoelectron microscopy, and to see whether any specific G alpha subunit was present in the mitotic spindle region. Subsequently, we used a colocalization method to identify the overlapping distributions of specific G alpha subunits and beta-tubulin. To be sure that the findings represented a general phenomenon, several other cell types were also examined. To identify whether the G alpha subunit in the mitotic spindle was present in a heterotrimeric form, we performed ADP ribosylation experiments. RESULTS: (a) Only Go alpha subunit but not Gi alpha or Gs alpha was localized in the mitotic spindle. (b) Go alpha was colocalized with beta-tubulin in the mitotic spindle. (c) The precise site of localization of Go alpha on spindle microtubules was confirmed by immunoultrastructural study. (d) The association of Go alpha with the mitotic spindle could be found in both human and animal cell types. (e) The mitotic spindle preparation could be ADP ribosylated in the presence of pertussis toxin. CONCLUSIONS: The heterotrimeric Go protein is shown to be closely associated with the mitotic spindle. This finding suggests that Go protein may play some role in the regulation of mitotic spindles in addition to its regulation of signal transduction in neuronal cell membranes.

Adenosine Diphosphate Ribose↗
PubMed1994

Comparison of five modalities in localization of primary aldosteronism.

BACKGROUND: The best response to surgical treatment of primary aldosteronism occurs in patients with an aldosterone-producing adenoma (APA), so pre-operative localization of the lesion is necessary. METHODS: A comparison was made of five localizing modalities-computed tomography (CT) of the adrenal gland, magnetic resonance imaging (MRI), dexamethasone suppression-131I-19-cholesterol adrenal scintiscan (DS, NP-59 adrenal scan), adrenal venography (venography) and adrenal venous sampling for aldosterone content (AVS) in 22 patients (12 women, 10 men) with primary aldosteronism; all had undergone operative confirmation within the past 9 years. The age at diagnosis ranged from 27 to 67 years (mean: 39.7). RESULTS: Unilateral adrenalectomy resulted in normal blood pressure without medication in 63.6% (14/22), and in improvement in 36.4% (8/22). Correct localization of the lesion was obtained in 95% (20/21) by CT, 100% (7/7) by MRI, 80% (12/15) by DS,NP-59 adrenal scan, 100% (6/6) by AVS and 78% (7/9) by venography. CONCLUSIONS: This experience would advocate CT of the adrenals as the initial means of localizing an APA on an outpatient basis because it is comfortable, safe, inexpensive and gives immediate results. The major role of MRI in the evaluation of adrenal adenoma should be complementary to CT. DS, NP-59 adrenal scan can be an adjuvant method for localization if CT scan results are not definitive. Adrenal venous catheterization with blood sampling for aldosterone content could be reserved for patients whose biochemical finging suggests the presence of an APA, and for whom CT or MRI of the adrenals and DS, NP-59 adrenal scan are inconclusive.

Adrenal Glands↗
PubMed1993

Epidermal growth factor receptor-mediated autocrine and paracrine stimulation of human transitional cell carcinoma.

Stromal-epithelial interactions may play a key role in tumor growth and metastasis. We have established a model to study the cellular and molecular basis of this paracrine interaction both in vivo and in vitro using a human transitional cell carcinoma cell line (WH). s.c. coinoculation of 1 x 10(6) WH cells with 1 x 10(6) nontumorigenic fetal rat urogenital sinus mesenchymal (rUGM) cells in athymic mice accelerated carcinoma growth 20 times faster than isolated WH cell inoculations and 4 times faster than coinoculations of the same number of NIH-3T3 or human bladder fibroblasts. Characterization of these chimeric tumors with immunohistochemical and DNA dot-blot analyses documented their predominantly human component. To evaluate the underlying mechanisms involved in this paracrine-mediated in vivo tumor growth acceleration, Northern analyses for growth factors (GFs) and extracellular matrix (ECM) expression in the different cell lines, as well as in vitro mitogenic assays, were performed. Northern analysis revealed basic fibroblast growth factor, transforming growth factor alpha, and epidermal growth factor receptor expression by WH cells but not rUGM cells; ECM components (fibronectin and collagens I and IV) were expressed only in the fibroblast cell lines. Cell type-specific paracrine growth factors are produced by cultured stromal and epithelial cells with a 2-3-fold bidirectional increase in WH and rUGM cell growth when cultured with reciprocal cell-type conditioned medium. An autocrine growth loop was observed for WH but not rUGM cells. WH cell growth is stimulated in vitro by low concentrations of transforming growth factor alpha and epidermal growth factor, while rUGM cell growth is stimulated 3-fold by basic fibroblast growth factor. Antiepidermal growth factor receptor antibodies completely inhibited autocrine and paracrine pathways stimulating WH cell growth, while anti-basic fibroblast growth factor antibodies had no inhibitory effect. These observations suggest that autocrine and paracrine growth factor stimulation of WH bladder carcinoma cell growth is most likely mediated by an epidermal growth factor receptor-related pathway. The predominant expression of ECM by fibroblasts in this model suggests that stromal cell ECM components may modulate tumor cell growth and angiogenesis possibly through mechanisms involving cellular adhesion, chemotaxis, or growth factor action.

3T3 Cells↗