[The expression of GGCT in the bladder urothelial cell carcinoma and its clinical significance].

Objective: To evaluate the expression and clinical significance of γ-glutamylcyclotransferase (GGCT) in patients with bladder urothelial cell carcinoma. Methods: Immunohistochemical staining for GGCT were performed on tissue sections of 86 patients with bladder urothelial cell carcinoma and 10 normal controls, and the correlations between GGCT and clinicopathological characteristics and the prognosis were analyzed. Results: The positive rate of the expression of GGCT in 86 cases of bladder urothelial cell carcinoma was 61.6% (53/86). GGCT protein was located mainly in cancer cell cytoplasm, and it can be seen in the nucleus of the tumor cells in some cases. The level of GGCT expression was positively related to pathological classification (P<0.001), stage (P=0.020), and tumor size (P=0.025). Immunohistochemical semiquantitative analysis showed that the expression of GGCT in patients with T1 stage of non-muscle invasion bladder urothelial cell carcinoma was significantly higher than that with Ta stage (P=0.034). Kaplan-Meier analysis showed that the expression of GGCT was correlated with the recurrence-free survival in patients with non-muscle invasive bladder cancer, the recurrence-free survival rate was lower in the GGCT positive group (P=0.029). Multivariate COX regression analysis showed that the pathological stage (OR=5.029, P=0.009) and the number of tumors (OR=3.320, P=0.024)were the independent risk factors for recurrence-free survival in patients with early urothelial cell carcinoma of the bladder. Conclusions: The expression of GGCT is significantly increased in bladder urothelial cell carcinoma and is related to the malignant biological behavior and progression of tumor. Patients with GGCT positive early bladder tumor are inclined to recur.

Identification of low potassium stress-responsive proteins in tobacco (Nicotiana tabacum) seedling roots using an iTRAQ-based analysis.

Potassium is one of the three main mineral nutrients, and is vital for leaf growth and the quality of tobacco (Nicotiana tabacum) plants. In recent years, the isobaric tags for relative and absolute quantitation (iTRAQ) method has been one of the most popular techniques for quantitative proteomic analysis. In this study, we used iTRAQ to compare protein abundances in the roots of control and low potassium-treated tobacco seedlings, and found that 108 proteins were differentially expressed between the two treatments. Of these, 34 were upregulated and 74 were downregulated, and 39 (36%) were in the chloroplasts. Kyoto Encyclopedia of Genes and Genomes pathway enrichment results suggested that metabolic pathways were the dominant pathways (10 upregulated and 14 downregulated proteins). Ten proteins involved in the pyruvate metabolism pathway increased their expression levels, and 17 upregulated proteins were enriched in the ribosomes category. To evaluate correlations between protein and gene transcript abundances, the expression patterns of 12 randomly chosen genes were examined. A quantitative real-time polymerase chain reaction revealed that the 12 genes were induced after low potassium treatment for 3, 6, 12, and 24 h. Our results demonstrate that low potassium levels affect protein profiles in tobacco roots.

[Clinical characters of pre-malignant and malignant polyp in postmenopausal women and the diagnostic value of hysteroscopy].

OBJECTIVE: To investigate the clinical characteristics of pre-malignant and malignant polyps in postmenopausal patients and to evaluate the diagnositic value of hysteroscopy in this disease. METHODS: From June 2005 to October 2014, 403 postmenopausal patients with polyps were treated in the Gynecologic Minimally Invasive Center, Beijing Obstetrics and Gynecology Hospital. There were 27 patients including 22 patients with pre-malignant and 5 patients with malignant polyps. All malignant lesions were endometrioid adenocarcinoma. The clinical data were retrospectively analyzed. Based on pathology, the diagnostic value of hysteroscopy was evaluated. RESULTS: (1) CLINICAL CHARACTERISTICS: there were 27 cases with pre-malignant and malignant polyps (group 1) and 376 cases with benign polyps (group 2). Compared the two groups, the average age was (60±8) vs (58±6) years old, the period of menopause was (9.8±8.1) vs (8.3±6.9) years. Thirteen cases (48.1%, 13/27) in group 1 and 159 cases (42.3%, 159/376) in group 2 had clinical symptoms including postmenopausal bleeding and vaginal discharge. Twelve cases (44.4%, 12/27) in group 1 and 140 cases (37.2%, 140/376) in group 2 were with hypertension. Five cases (18.5%, 5/27) in group 1 and 43 cases (11.4%, 43/376) in group 2 were with diabetes. The measures above were no significant differences (P>0.05) (2) Ultrasound features: the average thick of endometrium in group 1 and group 2 were respectively (1.3±0.7) and (0.8±0.4) cm, which had statistical significance (t=4.98, P=0.001). (3) Hysteroscopic diagnosis: the average diameters of polyp in group 1 and group 2 were respectively (2.4±1.0) and (1.6±1.0) cm, which had statistical significance (t=2.93, P=0.004). Six cases in group 1 were diagnosed by hysteroscopy including 4 cases of malignant polyp and 2 cases of pre-malignant polyp. The sensitivity, specificity, positive predictive value, negative predictive value and the accuracy were 22.2% (6/27), 100.0% (376/376), 100.0% (6/6), 94.7% (376/397) and 94.8% (382/403), respectively. CONCLUSIONS: Pre-malignant and malignant endometrial polyps are more common in the subjects with the larger diameters and the thicker endometrium. All polyps should be under complete resection by hysteroscopy and through pathology examination.

Expression of recombinant BMP-7 gene increased ossification activity in the rabbit bone mesenchymal stem cells.

OBJECTIVE: The mesenchymal stem cells (MSCs), which were distributed in the bone marrow stroma, become ideal progenitor cells in bone tissue engineering because of their convenient isolation, small injury when obtained, and strong osteogenic capacity. The osteogenic differentiation of MSCs, which is indicated by the increased alkaline phosphatase (ALP) activity and the enhanced accumulation of collagen, could be induced by a strong osteogenic capacity biological factor termed bone morphogenetic protein-7 (BMP-7). Although the chemically synthesized BMP-7 was widely applied to study the osteogenic differentiation of MSCs, transferring and expressing BMP-7 gene in target cells is more desirable, especially for gene therapy, given the advantages and convenience on the stable expression of BMP-7. The aim of this study was to determine whether recombinant BMP-7-expressing MSCs would induce bone formation in vitro. MATERIALS AND METHODS: BMP-7 gene was cloned from human placental tissue to construct a recombinant eukaryotic expression plasmid carrying BMP-7 gene by conjugating with eukaryotic expression vector pcDNA3.1. MSCs were isolated from rabbit bone marrow and cultured in vitro. Then they were divided into 3 groups: pcDNA3.1-BMP-7-transfected, pcDNA3.1-transfected, and untransfected. Human healthy fresh placental tissue was provided by the Department of Gynaecology and Obstetrics, Second Affiliated Hospital of Harbin Medical University. Written informed consent was obtained from the women. One healthy male New Zealand rabbit was provided by the Laboratory Animal Center, Harbin Medical University. RESULTS: A significant increase of ALP activity was detected in the supernatant of pcDNA3.1-BMP-7 transfected MSCs, and the enhanced collagen accumulation, which was inferred by the increased hydroxyproline content and RT-PCR. CONCLUSIONS: These results implied that BMP-7 gene was expressed in MSCs sufficiently and was involved in inducing differentiation of MSCs into osteoblast.

Cladosporium cladosporioides Identified in China on Tobacco Seeds.

Tobacco (Nicotiana tabacum L.) is a leafy, annual, solanaceous plant grown commercially for its leaves. China is the biggest single tobacco market and accounts for more than 40% of the global tobacco consumption (1). Tobacco seed harvested in Guiyang, Guizhou Province, China, are commonly contaminated or infected by various fungal pathogens, which can cause abnormal seedlings with dark brown lesions and stunting of roots and decayed seeds. In 2013, five samples of 500 seeds from tobacco cv. Guiyan 4 were tested for germination on moistened paper on petri dishes. On average, 35% of the seeds from all five samples developed into abnormal seedlings or were decayed and were plated onto potato dextrose agar media and grown for 5 days at 25°C in darkness to confirm the presence of a pathogen. However, one fungus was isolated from an average of 10% of the 500 seeds sampled. It was identified morphologically as Cladosporium cladosporioides (Fresen.) de Vries based on the velvety olive-brown with almost black reverse colony color and dimensions and color of conidia and conidiophores. Conidia formed in long branched chains that readily disarticulate, single celled, elliptical to limoniform, 2 to 8 (avg. 4.3) × 2 to 3 (avg. 2.1) µm. Conidia were pale to olive brown and smooth to verruculose. Ramoconidia were 0 to 1 septate, 7 to 14 (avg. 9.2) × 2 to 4 (avg. 2.6) µm, smooth or sometimes minutely verruculose. Conidiophores were pale to olive brown, macro- and micronemateus, smooth or sometimes verruculose, and of various lengths up to 320 µm long and 2 to 5 µm wide. Primer pair ITS1 and ITS4 was employed to amplify the regions of ITS1-5.8s-ITS2 of the pathogens. Sequences of all three isolates (G3, G10, and G18) (Accession Nos. KF841547, KF841554, and KF841560) were identical to each other and to four sequences in GenBank (JX230994.1, JQ768317.1, JQ768322.1, and AB763555.1). Pathogenicity of the three isolates of C. cladosporioides was verified on tobacco seedlings of 3-week-old grown on wet filter paper in the petri dishes (9 cm in diameter). For each isolate, 20 seedlings incubated in one plate were inoculated with 0.5 ml of a suspension of 105 conidia/ml. Twenty seedlings were treated with sterile water as control treatment. After inoculation, the petri dishes were incubated at 25°C, 100 to 120 µEm-2 S-1, RH > 80%, and 16 h light per day for disease development. At 96 h after inoculation, symptoms comprising medium brown to black lesions on the roots were clearly visible on inoculated plants but not on the control plants. All seedlings inoculated died 9 days after inoculation whereas control seedlings remained symptomless. Re-isolation attempts on PDA from roots demonstrated C. cladosporioides to be present in symptomatic seedlings but not in roots of the control plants. Moreover, the characteristics of the cultured fungi were exactly the same as those originally isolated. Isolates G3, G10, and G18 (KF841547, KF841554, and KF841560) were deposited with the Tobacco Diseased Fungi, Guizhou Academy of Tobacco Sciences, Guizhou, China. Previously, C. cladosporioides has also been isolated from macadamia (Macadamia integrifolia Maiden & Betche) racemes in South Africa (4), from diseased papaya (Carica papaya L.) in Taiwan province of China (2), and from seeds of Amaranthus spp. in Poland (3). To the best of our knowledge, this is the first report of C. cladosporioides causing seed disease on tobacco in China and the disease should be considered in existing disease management practices. References: (1) British American Tobacco Annual Report, 8, 2012. (2) R. S. Chen, et al. Plant Dis. 93:426, 2009. (3) W. Pusz. Phytopathologia 54:15, 2009. (4) N. van den Berg et al. Plant Dis. 92:484, 2008.

A comparative study of locoregionally advanced nasopharyngeal carcinoma treated with intensity modulated irradiation and platinum-based chemotherapy.

PURPOSE: To investigate the prognosis of three subgroups of locoregionally advanced nasopharyngeal carcinoma treated with intensity-modulated radiotherapy and platinum-based chemotherapy. PATIENTS AND METHODS: Hundred and eighty-one consecutive patients with locoregionally advanced untreated nasopharyngeal carcinoma were retrospectively divided into three subgroups: locally advanced group (T3-4N0-1M0), regionally advanced group (T1-2N2-3M0) and the mixed group (T3-4N2-3M0). They were all treated with definitive intensity-modulated radiotherapy and platinum-based chemotherapy. Their prognosis were investigated and compared. Multivariate analysis was applied to identify the independent risk factors of study endpoints. RESULTS: The 3-year locoregional control rates for locally advanced group, regionally advanced group, and the mixed group were 91.5%, 90.6% and 84.3% respectively, no significant difference was observed (P=0.656, P=0.429). The 3-year distant metastasis-free survival rates were 89.6%, 75.7% and 76.3%, respectively. The distant metastasis-free survival rate of the locally advanced group was significantly higher than the other two subgroups (P=0.028, P=0.028). The 3-year progression-free survival rates were 85.5%, 67.9% and 67.1% respectively with significance also favoring the locally advanced group (P=0.043, P=0.023). Nodal stage and the performance status were the independent risk factors of distant metastasis in the observed period. CONCLUSIONS: In the context of intensity-modulated radiotherapy and platinum-based chemotherapy, the locally advanced group had a better prognosis compared with the regionally advanced group and the mixed group. Treatment stratification may be based on nodal stage.

First Report of Fusarium Wilt of Tobacco Caused by Fusarium kyushuense in China.

Tobacco (Nicotiana tabacum L.) is a leafy, annual, solanaceous plant grown commercially for its leaves. China accounts for more than 39.6% of total global tobacco production (3). In May 2012, seedlings of tobacco cv. Honghuadajinyuan in a Guiyang tobacco commercial field (Guizhou, China, 26.35° N, 106.42° E) developed symptoms of severe wilting, chlorosis, and stunting. The main stem and taproot exhibited reddish to light brown vascular discoloration; further progression of these symptoms eventually caused mortality of infected seedlings. To isolate the causal agent, necrotic tissues from the symptomatic root were placed on potato dextrose agar (PDA) and incubated at 25°C in darkness. Colonies with white to rose mycelia and red-brown colony colors developed on PDA after 5 days of incubation. Microconidia were abundant, straight or slightly curved, clavate, 0- to 3-septate, and 7.5 to 20.0 × 2.5 to 5.0 µm. Macroconidia were straight or slightly curved, slender, 3- to 5-septate, and 25.0 to 45.0 × 3.3 to 5.0 µm. Based on the observed colony attributes, growth patterns, absence of chlamydospores, micro- and macro-spore attributes (1), and PCR amplification (using primers ITS1/4) combined with translation elongation factor primers (EF1/2) (2), the fungus was identified as F. kyushuense O'Donnell & T. Aoki. Sequence of ITS1-5.8s-ITS2 region of rDNA (GenBank Accession No. JX235957) exactly matched the sequences of F. kyushuense accession AB587020.1 (100% similarity). Analysis of the elongation factor (EF-1alpha) gene of the fungus (JX658565) resulted in a 99% match for F. kyushuense accession AB674297.1. Pathogenicity of the fungus was confirmed by performing Koch's postulate as follows. Pure cultures of the fungus F. kyushuense obtained from symptomatic tissues of tobacco seedlings were grown on PDA for 6 days. Tobacco plants to be used in pathogenicity tests were germinated and grown on potting soils in a plastic container. Additional fertilization was supplied by adding 0.2 g/L of 20-20-20 (N-P-K) in the float water. When seedlings got 6-leaf stage, they were ready for pathogenicity tests. Spores harvested from these culture plates were suspended in sterile distilled water, adjusted to a concentration of 1 × 104 conidia/ml, and inoculated by irrigating 10 ml of the conidia suspension onto roots of each of the 12 tobacco seedlings with 6-leaf stage. A group of 12 seedlings of the same age treated with sterile water served as control. Inoculated seedlings were maintained at 25°C, 100 µE m-2.s-1, relative humidity >70%, and 16 h light per day, and monitored for 9 days for symptom development. Seedlings inoculated with conidia developed disease symptoms with roots with vascular discoloration of roots whereas control seedlings remained symptomless. F. kyushuense was reisolated from the symptomatic seedlings 9 days after inoculation. F. kyushuense has also been isolated from rice seeds in China (4), and from diseased wheat in Japan (1). The common tobacco Fusarium disease reported in China was caused by F. oxysporium f. sp. nicotianae. However, to the best of our knowledge, this is the first report of F. kyushuense causing wilt on tobacco in China and the disease must be considered in existing disease management practices. References: (1) T. Aoki and K. O'Donnell. Mycoscience. 39:1, 1998. (2) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (3) US Census Bureau. Foreign Trade Statistics. Washington DC, 2005. (4) Z. H. Zhao and G. Z. Lu. Mycotaxon. 102:119, 2007.

CD147 induces UPR to inhibit apoptosis and chemosensitivity by increasing the transcription of Bip in hepatocellular carcinoma.

The unfolded protein response (UPR) is generally activated in solid tumors and results in tumor cell anti-apoptosis and drug resistance. However, tumor-specific UPR transducers are largely unknown. In the present study, we identified CD147, a cancer biomarker, as an UPR inducer in hepatocellular carcinoma (HCC). The expression of the major UPR target, Bip, was found to be positively associated with CD147 in human hepatoma tissues. By phosphorylating FAK and Src, CD147-enhanced TFII-I tyrosine phosphorylation at Tyr248. CD147 also induced p-TFII-I nuclear localization and binding to the Bip promoter where endoplasmic reticulum (ER) stress response element 1 (ERSE1) (-82/-50) is the most efficient target of the three ERSEs, thus increasing transcription of Bip. Furthermore, by inducing UPR, CD147 inhibited HCC cell apoptosis and decreased cell Adriamycin chemosensitivity, thus decreasing the survival rate of hepatoma-bearing nude mice. Together, these results reveal pivotal roles for CD147 in modulating the UPR in HCC and raise the possibility that CD147 is a target that promotes HCC cell apoptosis and increases the sensitivity of tumors to anti-cancer drugs. Therefore, CD147 inhibition provides an opportunity to enhance the efficacy of existing agents and represents a novel target for HCC treatment.

Activator protein-1 transcription factor mediates bombesin-stimulated cyclooxygenase-2 expression in intestinal epithelial cells.

Colorectal carcinogenesis is a complex, multistep process involving genetic alterations and progressive changes in signaling pathways regulating intestinal epithelial cell proliferation, differentiation, and apoptosis. Although cyclooxygenase-2 (COX-2), gastrin-releasing peptide (GRP), and its receptor, GRP-R, are not normally expressed by the epithelial cells lining the human colon, the levels of all three proteins are aberrantly overexpressed in premalignant adenomatous polyps and colorectal carcinomas of humans. Overexpression of these proteins is associated with altered epithelial cell growth, adhesion, and tumor cell invasiveness, both in vitro and in vivo; however, a mechanistic link between GRP-R-mediated signaling pathways and increased COX-2 overexpression has not been established. We report that bombesin, a homolog of GRP, potently stimulates the expression of COX-2 mRNA and protein as well as the release of prostaglandin E(2) from a rat intestinal epithelial cell line engineered to express GRP-R. Bombesin stimulation of COX-2 expression requires an increase in [Ca(2+)](i), activation of extracellular signal-regulated kinase (ERK)-1 and -2 and p38(MAPK), and increased activation and expression of the transcription factors Elk-1, ATF-2, c-Fos, and c-Jun. These data suggest that the expression of GRP-R in intestinal epithelial cells may play a role in carcinogenesis by stimulating COX-2 overexpression through an activator protein-1-dependent pathway.

Hypersulfated low molecular weight heparin with reduced affinity for antithrombin acts as an anticoagulant by inhibiting intrinsic tenase and prothrombinase.

In buffer systems, heparin and low molecular weight heparin (LMWH) directly inhibit the intrinsic factor X-activating complex (intrinsic tenase) but have no effect on the prothrombin-activating complex (prothrombinase). Although chemical modification of LMWH, to lower its affinity for antithrombin (LA-LMWH) has no effect on its ability to inhibit intrinsic tenase, N-desulfation of LMWH reduces its activity 12-fold. To further explore the role of sulfation, hypersulfated LA-LMWH was synthesized (sLA-LMWH). sLA-LMWH is not only a 32-fold more potent inhibitor of intrinsic tenase than LA-LMWH; it also acquires prothrombinase inhibitory activity. A direct correlation between the extent of sulfation of LA-LMWH and its inhibitory activity against intrinsic tenase and prothrombinase is observed. In plasma-based assays of tenase and prothrombinase, sLA-LMWH produces similar prolongation of clotting times in plasma depleted of antithrombin and/or heparin cofactor II as it does in control plasma. In contrast, heparin has no effect in antithrombin-depleted plasma. When the effect of sLA-LMWH on various components of tenase and prothrombinase was examined, its inhibitory activity was found to be cofactor-dependent (factors Va and VIIIa) and phospholipid-independent. These studies reveal that sLA-LMWH acts as a potent antithrombin-independent inhibitor of coagulation by attenuating intrinsic tenase and prothrombinase.

Roles of gastrointestinal hormones in pancreatic cancer.

Several gastrointestinal (GI) hormones, such as gastrin, cholecystokinin, and bombesin, have been reported to affect the development of pancreatic cancer. The receptors for these hormones are found in normal and neoplastic pancreatic cells. Activation of these receptors enhances pancreatic carcinogenesis and promotes the growth of established pancreatic carcinoma either in vitro or in vivo. On the other hand, some studies have shown that these GI hormones may have no effect or may play an inhibitory role in the development of pancreatic cancer. The reasons for the apparent discrepancies in the published literature are discussed in this review. In recent years, increasing emphasis has been placed on the effects of GI hormones on cancer invasion and metastasis. As the transition from noninvasion to the invasive state is the crucial event in cancer development, further investigation of the way in which GI hormones affect the invasion and metastasis of pancreatic cancer may be important for the development of new therapeutic approaches with eventual clinical utility.

Insulin-like growth factor-I promotes multidrug resistance in MCLM colon cancer cells.

Insulin-like growth factor-I (IGF-I) is known as a potent mitogen for a variety of cell types, including colon cancer cell lines. The objective of this study was to determine the effect of IGF-I on cell death induced by cytotoxic agents actinomycin D (Act-D), lovastatin (LOV), and doxorubicin (DOX) in the MCLM mouse colon cancer cell line, and the mechanisms involved. Subconfluent monolayer MCLM cells were treated with IGF-I (25 ng/ml) for 12 h in serum-free media. Various concentrations of cytotoxic agents then were added to the cells that were incubated continually at 37 degrees C for 24 h. Cell survival was determined with the MTT (3-[4-5-dimenthylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, which assesses mitochondrial function in living cells. The mRNA expression for multidrug resistance gene-1 (mdr-1), c-H-ras, and manganese superoxide dismutase (MnSOD) in cells treated with IGF-I was examined by Northern blot or RNase protection assays. The levels of p-glycoprotein, a drug efflux pump encoded by the mdr-1 gene, were assessed by Western immunoblotting. Results demonstrated that 1) IGF-I significantly inhibited the cell death and apoptosis of MCLM cells treated with Act-D, LOV, or DOX; 2) IGF-I increased mRNA expression for mdr-1, c-H-ras, and MnSOD; 3) the p-glycoproteins in cells treated with IGF-I or stably transfected with c-H-ras were elevated when compared with control. These results suggest that IGF-I protects MCLM cells against death induced by cytotoxic agents; this acquired drug resistance may be mediated by multiple mechanisms, including promoting expression of mdr-1, c-H-ras, and MnSOD; whereas, the p-glycoprotein level stimulated by IGF-I may result partly from the increase of c-H-ras in the cells.

Regulation of growth of human gastric cancer by gastrin and glycine-extended progastrin.

BACKGROUND & AIMS: Gastrin (G-17) stimulates the growth of certain gastric and colon cancers mostly through gastrin/cholecystokinin (CCK)-B receptors. Glycine-extended gastrin (Gly-G) stimulates growth of a rat pancreatic acinar cell line; however, the effect of Gly-G on human gastric cancers is not known. The purpose of this study was to characterize the trophic effect of G-17 and Gly-G on two human gastric cancer cell lines, AGS and SIIA. METHODS: Binding analyses were performed, and cell growth was assessed by counting cells over a time course. RESULTS: G-17 stimulated growth of both AGS and SIIA cells. In AGS cells, gastrin/CCK-B receptor antagonists inhibited the effect of G-17 and competitively antagonized 125I-G-17 binding, whereas the CCK-preferring (CCK-A) receptor antagonists had no effect. In contrast, CCK-A receptor antagonists inhibited the stimulatory effect of G-17 in SIIA cells, whereas CCK-B receptor antagonists had no effect. Gly-G stimulated the growth of AGS and SIIA cells; neither the CCK-B nor the CCK-A receptor antagonists blocked this effect. CONCLUSIONS: G-17 stimulates proliferation of AGS cells through the CCK-B receptor; however, G-17-mediated growth of SIIA acts through a CCK-A-like receptor. Furthermore, Gly-G stimulates growth of human gastric cancer cell lines, possibly through a receptor other than the CCK-B or CCK-A receptor.

Insulin-like growth factor-II expression in carcinoma in colon cell lines: implications for autocrine actions.

BACKGROUND: In the gastrointestinal tract, insulin-like growth factor-II (IGF-II) messenger RNA (mRNA) is localized mainly in mesenchymal cells, and is more abundant in the fetus than in the adult. The purposes of this study are to characterize the gene expression of IGF-II at the mRNA and protein level in seven different epithelial cell lines derived from colon carcinomas and to determine the action of IGF-II and IGF-receptors on a colon carcinoma cell line. STUDY DESIGN: Insulin-like growth factor-II mRNAs were examined by Northern analysis; conditioned media from colon carcinoma cells were concentrated, chromatographed, and examined by a specific IGF-II radioreceptor assay. Insulin-like growth factor receptors were examined by radioligand binding assays. The mitogenic role of IGF-II was determined by a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay. RESULTS: Multiple sizes of IGF-II mRNAs were expressed in all colon carcinoma cell lines tested (six human cell lines: HCT116, COLO 205, COLO 320 DM, LoVo, DLD-1, and HT29, and one mouse cell line: MC-26). In the conditioned media of COLO 205 and HCT116 cells, 7.5 kilodaltons IGF-II and high molecular form (IGF-II and IGF binding protein complex) were detected. Both Type I and Type II IGF receptors were present on COLO 205 cells whose growth was stimulated by IGF-II. Addition of anti-IGF-I receptor and anti-IGF-II antibody in the cell culture significantly depressed growth of the COLO 205 cell line in the presence or absence of exogenous IGF-II. CONCLUSIONS: Insulin-like growth factor-II mRNAs are expressed in human and mouse colon carcinoma cell lines, which may induce production of a significant amount of biologically active IGF-II protein. The IGF-II secreted by COLO 205 cells may stimulate cell growth in an autocrine fashion through the Type I IGF receptors.

A study of the cytotoxicity of malignant pleural effusion lymphocytes and LAK cells against autologous tumor cells.

The cytotoxicity of malignant pleural effusion lymphocytes (MPEL) against autologous tumor cells (ATC) were compared with that of peripheral blood lymphocytes (PBL). It was demonstrated that the cytotoxicity of PBL was higher than that of MPEL (P < 0.001), but the cytotoxicity and expansion of MPEL-activated by rIL-2 was much higher than that of PBL activated by rIL-2 (LAK cells) (P < 0.001). This shows that local immune reaction of the pleural cavity of patients with malignant pleural effusion was in the state of suppression. MPEL activated are better effector cells than LAK cells in tumor adoptive immunotherapy.

In vivo mitogenic effects of estradiol on colon cancers: role of gastrin and gastrin receptors.

We have previously reported mitogenic effects of gastrin on a mouse colon cancer (MC-26) cell line in vivo. The present studies were undertaken to determine if gonadal hormones can influence the mitogenic response of MC-26 cells to gastrin. The female gonadal hormone, estradiol (E2), was determined to be as mitogenic as pentagastrin (PG) for the growth of MC-26 tumors in mice; the mitogenic effects of E2 and PG were not additive. Female gonadal hormones were furthermore as effective as PG in maximally up-regulating gastrin receptor (GR) concentrations on MC-26 tumor membranes, which was confirmed in autoradiographic studies. Since PG and E2 had similar and non-additive trophic effects it was hypothesized that gastrin may be mediating the trophic effects of E2. Serum gastrin concentrations were significantly increased in E2 treated ovariectomized mice that correlated with an increase in tumor weights; E2 however was ineffective in stimulating the release of gastrin from perfused rat stomachs indicating that the increase in serum gastrin concentration on long-term treatment with E2 was mediated by some other mechanism. Saturable high-affinity E2 binding sites were not measured in MC-26 cells and tumors, supporting the possibility that mitogenic effects of E2 were probably mediated via indirect mechanisms. In summary our results indicate that both E2 and PG are equally mitogenic for colon cancer cells in vivo which may explain the sex- and age-related discrepancy in the incidence of human colon cancers.

Insulinlike growth factor-binding protein modulates the growth response to insulinlike growth factor 1 by human gastric cancer cells.

BACKGROUND: This study determined whether the resistance to the mitogenic effect of insulinlike growth factor 1 (IGF-1) in AGS (we found that IGF-1 had almost no effect on the growth of AGS) cells is caused by the absence of IGF-1 receptor on the cells or by the interference of endogenous IGFs and IGF-binding protein (IGFBP). METHODS: IGF-1 receptors were examined by radioligand binding assay. The protein in conditioned medium and the molecular weight of IGF-1 receptors on AGS cells were determined by affinity cross-linking with 125I-IGF-1 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Messenger RNAs for IGF-1, IGF-2, and IGFBP-4 were detected by Northern analysis. RESULTS: AGS cells possessed a single class of high-affinity binding sites for IGF-1 (dissociation constant [Kd], 0.51), with a binding capacity approximately 4 x 10(4) sites per cell. The size of the alpha subunit of IGF-1 receptors on cell membranes was approximately 130 kilodaltons. des (1-3) IGF-1, a truncated IGF-1 with very low affinity to IGFBPs, stimulated AGS cell growth in dose-dependent fashion. The medium conditioned by AGS cells contained IGFBPs of 27-32 and 37-42 kilodaltons. AGS cells expressed messenger (mRNA) RNAs for IGF-2 and IGFBP-4 but not for IGF-1, whereas another gastric carcinoma cell line (SIIA), whose growth is stimulated by IGF-1, expressed mRNA IGF-2 but did not express mRNA for IGF-1 or IGFBP-4: CONCLUSIONS: The relative absence of growth response of AGS cells to IGF-1 is due to the endogenously produced IGFBPs sequestering IGF-1 and preventing receptor interaction.