Establishment and characterization of an IGSK-2 cell line derived from ascitic fluid of recurrent hCG and somatostatin secreted adenocarcinoma of the stomach

We examined a 32-year-old Japanese man who was clinically diagnosed with gastric cancer, type 4, and histopathologically diagnosed with mucinous and poorly differentiated adenocarcinoma (mucinous > poorly) of the stomach. We successfully established and characterized a cell line (designated as IGSK-2) derived from the ascitic fluid of the patient with recurrent and cisplatin-resistant carcinoma. The IGSK-2 cells grew in multi-layered culture in culture dishes. The cells secreted 18 pg/mL somatostatin, 9.1 mIU/mL human chorionic gonadotrophin (hCG), 8000 U/mL carbohydrate antigen 19-9 and 410 ng/mL carcinoembryonic antigen over 4 days of culture. The population doubling time was approximately 83 h. The susceptibility test of anticancer drugs revealed that IGSK-2 cells were sensitive to Taxol, but were not sensitive to cisplatin, 5-fluorouracil and irinotecan. Immunohistochemical staining revealed that the IGSK-2 cells were positive against antihCG antibody and antiserotonin antibody, and negative against antisomatostatin antibody and antigastrin antibody.     

Establishment and characterization of a cisplatin-resistant cell line (IGSK-1) from a poorly differentiated gastric adenocarcinoma

We successfully established a spontaneously cisplatin-resistant tumor cell line (designated as IGSK-1) derived from original gastric carcinoma. The patient was a 75-year-old Japanese woman. The histopathological diagnosis was gastric poorly differentiated adenocarcinoma accompanied with metastatic foci in lymph nodes, pT3, N2 M0, stage IIIB. The IGSK-1 cells grew as adhesive and monolayered cultures on the bottom of dishes. The susceptibility of the IGSK-1 cells to anti-cancer drugs was examined using oxygen electrode apparatus (Daikin, Tsukuba, JPN), and the results suggested TXL was effective, and CDDP, CPT-11 and 5-FU were not effective. Gastrin and somatostatin secretions were confirmed by immunohistochemical staining and also radioimmunoassay. Immunohistochemistry and radioimmunoassay for serotonin suggested the IGSK-1 cells might incorporate serotonin from the growth media. Spontaneously cisplatin-resistant gastric carcinoma cell line secreted gastrin and somatostatin is very important material for chemotherapy.     

Tumor angiogenesis factors produced by cancer cells.

Tumor angiogenic activity from tumor angiogenesis factors (TAFs) produced by 25 cell lines was assayed onto chorioallantoic membranes (CAMs). Neovascularization occurred prominently in such cell lines, as HTBOA (poorly differentiated ovarian carcinoma), HUOCA-II (poorly differentiated clear cell adenocarcinoma), HWUA (poorly differentiated endometrial adenocarcinoma), HKUS (uterine cervical small cell carcinoma), and in HOTHC (anaplastic thyroid carcinoma). The cell lines which secreted TAF showed high heterotransplantability in nude mice and produced rapidly growing tumors which were rich in blood vessels. The TAFs polypeptides of 14,000 and 78,000 molecular weight, were extracted and purified from the conditioned medium of HUOCA-II or W3UF (sub-line of HUOCA-II) lines, respectively. TAFs at concentrations of 10 ng/ml and 100 ng/ml promoted proliferation of the endothelial cells and induced tube formation. Microsequencing analysis revealed that TAF of 78,000 molecular weight has sequence identity with human hepatocyte growth factor (hHGF).     

Establishment and characterization of a human colonic cancer cell line expressing carbohydrate antigen 19-9 and carcinoembryonic antigen]

Colonic cancer cell strain KE43 was established from a human colonic cancer diagnosed histologically as a predominantly well differentiated adenocarcinoma with minute foci of poorly differentiated adenocarcinoma. The well differentiated adenocarcinoma cell line was identified as the major morphological picture in xenografts of KE43 and 58 in nude mice, but this changed to poorly differentiated adenocarcinoma in passage 105. Doubling time of this cancer cell line was 22.5 hours in passage 105. The modal numbers of chromosomes were 41 and 76. Cancer cells could be heterotransplanted in 100% of the nude mice. The tumor cells produced and secreted CA19-9, CEA and Laminin into the spent medium. This cell line appears to provide a useful system for studying colonic cancer in vivo and in vitro.     

Establishment and characterization of a novel ovarian serous adenocarcinoma cell line, TU-OS-4, that overexpresses EGFR and HER2

A new line of human ovarian serous adenocarcinoma cells, TU-OS-4, was established and characterized. The cells showed a short, spindle-shaped morphology and grew in monolayers without contact inhibition while forming an arrangement resembling a jigsaw puzzle. Chromosome numbers ranged from 55 to 73. The proliferation rate was lower than other serous adenocarcinoma cell lines tested (KF, SHIN-3, and SK-OV-3), and the doubling time was 53.3 h. Western blot analysis showed that TU-OS-4 cells overexpressed epidermal growth factor receptor, human epidermal growth factor receptor (HER) 2, and phosphorylated HER2 protein. The IC₅₀ values to cisplatin, paclitaxel, and lapatinib were 25.8 μM, 686 nM, and 183 nM, respectively. Heterotransplantation in nude mice reflected the original tumor of the cells. These results suggested that this cell line would be useful to study chemoresistant mechanisms and contribute to establishing novel treatment strategies for patients with ovarian cancer.     

Production and utilization of growth factors related to fibroblast growth factor by embryonal carcinoma cells and their differentiated cells

Previous studies have established that embryonal carcinoma (EC) cells produce several different growth factors, but express few, if any, receptors for epidermal growth factor, platelet-derived growth factor, or transforming growth factor type-beta. In this study, the production and utilization of fibroblast growth factor (FGF) by EC cells and their differentiated cells were investigated. We have determined that EC cells produce a heat-labile, heparin-binding factor that competes with FGF for binding to membrane receptors and appears to be immunologically related to FGF. The same or a similar factor is produced by three different EC cell lines, including a multipotent human EC cell line. However, production of this factor is apparently reduced when each EC cell line differentiates. Unlike the parental EC cells, the differentiated cells respond to FGF by growth stimulation and the growth responses to FGF correlate with increased binding of FGF. Although the binding data indicate that both the EC cells and their differentiated cells exhibit high affinity receptors for FGF, the differentiated cells express these receptors at levels approximately 10-fold higher. These findings suggest that the FGF-related growth factor could influence the growth of EC cells or their differentiated cells.     

Effects of growth stimulatory factors on mitogenicity and c-myc expression in poorly differentiated and well differentiated human colon carcinoma cells

We demonstrate the differential sensitivity of poorly differentiated and well differentiated human colon carcinoma cells to nutrients alone or to nutrients and polypeptide growth factors under completely serum-free conditions. 3H-Thymidine incorporation into trichloroacetic acid precipitable material and autoradiographic analysis indicated that nutrient replenishment alone was sufficient to initiate DNA synthesis in quiescent poorly differentiated cells, whereas defined polypeptide growth factors produced no additional effect. In contrast, well differentiated cells were mitogenically stimulated to a much greater extent by growth factors (epidermal growth factor + insulin + transferrin), than by nutrient replenishment alone. Expression of the c-myc protooncogene was increased approximately 5-fold after growth factor addition to the well differentiated cells. Maximal expression of c-myc occurred at 4 h post stimulation. In contrast, nutrients resulted in only a slight up-regulation of c-myc (1.8-fold) at approximately 90 min after addition. Addition of nutrients and/or growth factors to the poorly differentiated colon carcinoma cells resulted in an initial decline in c-myc expression (90 min), presumably due to removal of endogenous growth stimulators. Expression of c-myc returned to baseline levels by 24 h after additions. The results indicate that differential sensitivity to polypeptide growth factors is related to differentiation status in this model system and suggest that the insensitivity of poorly differentiated cells to exogenous growth factors may be due to a greater production of autocrine growth stimulators.     

Parathyroid hormone-related protein is a possible autocrine growth inhibitor for lymphocytes

Adult T-cell leukemia (ATL)-related cells have the ability to produce a newly-isolated calcium-regulating protein, parathyroid hormone-related protein (PTHrP). The present study revealed that lectin-stimulated normal lymphocytes produce immunoreactive (IR)-PTHrP. When the T-cell-enriched fraction was purified from normal lymphocytes and then treated with lectin, a similar amount of IR-PTHrP was detected, suggesting that IR-PTHrP is an actual product of T-lymphocytes. A biologically active fragment of PTHrP, PTHrP(1-34), suppressed DNA synthesis in lectin-stimulated lymphocytes at concentrations greater than 50 pg/mL; the same concentration range of IR-PTHrP detected in the cultured media of lectin-stimulated lymphocytes. Therefore, it is reasonable to postulate that PTHrP is a cytokine inhibiting the cellular growth of normal lymphocytes.     

Expression of the pS2 gene in human gastric cancer cells derived from poorly differentiated adenocarcinoma

NH2-terminal amino acid sequence of the pS2 protein produced and secreted by human gastric cancer cells, MKN-45, was determined to be identical to that of MCF-7 cells. A clone encoding pS2 protein was isolated from the cDNA library constructed from MKN-45 cells. The nucleotide sequence was identical to that of pS2 cDNA previously isolated from human breast cancer cells, MCF-7, except for one nucleotide in the 3' untranslated region. Thus, in this cell line, the pS2 gene product is translated and secreted as in MCF-7 cells. RNA blot hybridization analysis revealed that pS2 gene was expressed well in two (MKN-45 and KATO-III; derived from poorly differentiated adenocarcinoma) but not in three cell lines (MKN-1, MKN-28 and MKN-74; from well differentiated adenocarcinoma), suggesting that expression of the pS2 gene depends on the state of cell differentiation. These results suggest that pS2 is expressed in human gastric cancer cells in an estrogen-independent manner and is possibly associated with the malignant state of cells.     

Establishment and characterization of a human chorionic gonadotropin (hCG) producing cell line (RTSG) from an ovarian epithelial cancer

A new cell line designated RTSG established in vitro from the pleural effusion of a patient with metastatic ovarian epithelial cancer has been subcultured 46 times for more than 2 years. The cells grew in a monolayered sheet, showing a tendency to pile up, with the population doubling in 48 hrs. Electron-microscopically, desmosomes were characteristically observed, suggesting the cells were of epithelial origin. Chromosomal analysis revealed aneuploidy with a tetraploid mode. The heterotransplanted tumors in nude mice were histopathologically classified as a poorly differentiated adenocarcinoma, whereas the original tumor consisted mainly of mucinous and serous cystadenocarcinoma and only partly of poorly differentiated adenocarcinoma. The cells secreted hCG (38.8 mIU/day/10(6) cells) and beta-hCG (6.1 ng/day/10(6) cells) in spent medium. Immunocytologic +-and-histochemical staining for tumor markers of the original tumor, the cultured cells and the transplanted tumors also revealed the localization of not only hCG and beta-hCG but also CA19-9 and CA-125 whose values had been elevated in the preoperative serum (hCG: 10 mIU/ml, CA19-9: 6,400 U/ml, CA-125: 225 U/ml). Results of PAS, Alcian-blue and Mucicarmine strains indicated that most of the PAS-positive substances in the cultured cells and the transplanted tumors were consistent with glycogen while the original tumor mainly contained mucin except for the lesion of poorly differentiated adenocarcinoma with glycogen. These results suggested that the cultured cells might originate from poorly differentiated adenocarcinoma cells in the original tumor.     

Clonal rat parathyroid cell line expresses a parathyroid hormone-related peptide but not parathyroid hormone itself

A novel parathyroid hormone-related peptide has been identified in tumors associated with the syndrome of humoral hypercalcemia of malignancy. Subsequently, mRNAs encoding this peptide have been found to be expressed in a number of normal tissues, including the parathyroids. Using Northern blotting, RNase protection, and immunochemical techniques, we examined a clonal rat parathyroid cell line originally developed as a model system for studying parathyroid cell physiology. We found that this line expresses the parathyroid hormone-related peptide but not parathyroid hormone itself. Secretion of the parathyroid hormone-related peptide varied inversely with extracellular calcium concentration, but neither calcium nor 1,25-dihydroxyvitamin D3 appeared to influence steady-state parathyroid hormone-related peptide mRNA levels. This clonal line may prove to be an interesting system for studying the factors responsible for tissue-specific parathyroid hormone and parathyroid hormone-related peptide gene expression.     

A new human cholangiocellular carcinoma cell line (HuCC-T1) producing carbohydrate antigen 19/9 in serum-free medium

Summary A human cholangiocellular carcinoma cell line, HuCC-T1, was established in vitro from the malignant cells of ascites of a 56-yr-old patient. Histologic findings of the primary liver tumor revealed a moderately differentiated adenocarcinoma. Tumor cells from the ascites have been cultured with RPMI 1640 medium containing 0.2% lactalbumin hydrolysate and the cultured cells grew as monolayers with a population doubling time of 74 h during exponential growth at Passage 25. They had an epithelial-like morphology and were positive for mucine staining. Ultrastructural studies revealed the presence of microvilli on the cell surface and poorly developed organelles in the cytoplasm. The HuCC-T1 cell was tumorigenic in nude mice. The number of chromosomes in HuCC-T1 ranged from 61 to 80. These human cholangiocellular carcinoma cells in serum-free medium secreted several tumor markers, including carbohydrate antigen 19/9, carbohydrate antigen 125, carcinoembryonic antigen, and tissue polypeptide antigen. The carbohydrate antigen 19/9 secretion level of HuCC-T1 cells cultured in PRMI 1640 medium with 1% fetal bovine serum was sixfold higher than that with 0.2% lactalbumin hydrolysate. These findings suggest that HuCC-T1 will provide useful information to clarify the mechanism of tumor marker secretion and tumor cell growth in the human cholangiocellular carcinoma.     

人CD137抗体促进NK细胞对乳腺癌细胞特异性杀伤作用的体外研究

目的探讨人自然杀伤(NK)细胞在CD137抗体作用下通过抗体依赖性细胞毒性作用(ADCC)介导对乳腺癌细胞的杀伤作用。方法NK细胞表型和细胞因子检测实验分组:阴性对照组(未用人CD137抗体处理的NK细胞)、CD137抗体处理组(10μg/mL人CD137抗体处理4 h的NK细胞);NK细胞毒性检测实验分组:根据体系中是否添加NK细胞、人CD137抗体和西妥昔单抗分为8组。流式细胞术检测两组NK细胞表面CD16分子的表达情况,酶联免疫吸附测定(ELISA)法检测两组NK细胞培养上清液中干扰素(IFN)-γ和肿瘤坏死因子(TNF)-α的浓度,乳酸脱氢酶(LDH)法检测8组反应体系中表皮生长因子受体(EGFR)高表达乳腺癌细胞系MDA-MB-231和EGFR低表达乳腺癌细胞系MDA-MB-453的杀伤比例,并采用t检验或析因分析进行统计学分析。结果与阴性对照组比较,CD137抗体处理组CD16+NK细胞比例(79.57﹪±0.92﹪比90.43﹪±0.67﹪)、细胞因子IFN-γ浓度[(388.90±7.02)pg/mL比(523.90±1.90)pg/mL]和TNF-α浓度[(20.59±4.09)pg/mL比(47.22±2.14)pg/mL]均升高,差异有统计学意义(P<0.05);MDA-MB-231细胞杀伤的三因素析因分析结果显示:NK细胞、人CD137抗体和西妥昔单抗3个因素分别对MDA-MB-231细胞的杀伤都有作用(F=5227.276、201.473、1792.242,P均<0.001),3个因素两两之间的交互作用对MDA-MB-231细胞的杀伤也都有作用(F=183.903、1517.187、33.483,P均<0.001),3个因素的二级交互作用差异有统计学意义(F=41.505,P<0.001)。结论人CD137抗体可增强NK细胞分泌细胞毒性因子IFN-γ和TNF-α的能力,同时可上调NK细胞表面CD16分子的表达,从而使得NK细胞可能通过西妥昔单抗介导的ADCC作用增强对表皮生长因子受体(EGFR)高表达乳腺癌细胞的杀伤作用。     

The effect of transforming growth factor beta on the intensity of cellular DNA synthesis in relation to the type and conditions of cultivation

A study was made of the influence of transforming growth factor beta (0.05-50.00 ng/ml) on the intensity of 3H-thymidine incorporation in the DNAs of pseudonormal mouse fibroblasts of NIH 3T3 line, of cells of NRK-49F line from normal rat kidney, and of cells of A-549 line from human lung adenocarcinoma. The experiments were carried out in the absence and in the presence of epidermal growth factor (5 ng/ml), and(or) insulin (1 micrograms/ml), as well as in the presence of different concentrations of fetal calf serum, and while using different time of incubation of cells with the above mentioned growth factors. It was shown that depending on the culture conditions the transforming growth factor beta exerted stimulatory, inhibitory or no action on the intensity of DNA synthesis in the cells of the same type. An attempt was made to analyse the reasons, which may significantly determine the direction of regulatory influence of the transforming growth factor beta on DNA replication in the cells.     

Functional parathyroid hormone receptors are present in an umbilical vein endothelial cell line

Acute parathyroid hormone exposure induces vascular smooth muscle relaxation. In contrast, continuous infusion of parathyroid hormone leads to vasoconstriction and an elevation of blood pressure. Despite the known effects of parathyroid hormone on vascular smooth muscle, possible direct effects on the vascular endothelium have not previously been investigated. Using a human umbilical vein endothelial cell line, we found that parathyroid hormone increased both intracellular calcium and cellular cAMP content in these endothelial cells. Furthermore, exposure of these cells to increasing concentrations of parathyroid hormone stimulated both [(3)H]thymidine incorporation and endothelin-1 secretion. Parathyroid hormone/parathyroid hormone-related peptide receptor mRNA could be detected at low levels in these cells. In summary, these data demonstrate that endothelium-derived cells contain functional parathyroid hormone receptors. The potential physiological role of these receptors remains to be determined.     

Coexpression of PTHrP and PTH/PTHrP receptor in a myoepithelial cell line derived from normal human breast

Parathyroid hormone-related peptide (PTHrP) is the cause of humoral hipercalcaemia of malignancy syndrome (HHM). It is known that the peptide as well as its receptors are widely distributed in many normal organs and tissues, where it influences an array of diverse functions which are realized through paracrine or autocrine pathway. PTHrP is present in large amounts in lactating mammary gland but its function is not fully elucidated. In this study, production of parathyroid hormone-related peptide (PTHrP) by the Hs578Bst cell line corresponding to mammary myoepithelial cells was examined by immunocytochemistry. Using RNA extracted from these cells we analyzed expression of mRNA for PTHrP and for the PTH/PTHrP receptor by RT-PCR. The obtained results demonstrated that Hs578Bst cells produced PTHrP and synthesized mRNA for PTHrP and PTH/PTHrP type I receptor. It provides evidence that myoepithelial cells are target cells for PTHrP. The data support that PTHrP may be an important autocrine/paracrine factor, involved in the regulation of myoepithelial cell function as well as in growth and differentiation of the mammary gland.     

A cell culture model of chemically and spontaneously derived mouse lung alveologenic carcinoma

Malignant cell lines related to mouse lung alveologenic carcinoma have been established from urethane-induced tumors and after in vitro spontaneous transformation of preneoplastic cell lines. Both the chemically and spontaneously transformed cell lines formed invasive, poorly differentiated carcinomas with secondary lung deposits when implanted subcutaneously in immune-suppressed mice. They differed from the related preneoplastic cell line in coordinately exhibiting anchorage-independent growth, reduced epidermal growth factor receptor activity and absence of pericellular fibronectin. These data suggest that similar molecular events may occur in type 2 pneumocyte-related cells in order to generate mouse lung alveologenic adenomas and carcinomas by both spontaneous and chemical carcinogen induction mechanisms. A reduced level of pericellular fibronectin was also demonstrated in an in situ compressive urethane-induced mouse lung adenoma. Loss of pericellular fibronectin may therefore be an early and persistent phenotypic alteration during transformation to the alveologenic adenoma and carcinoma.Abbreviations EGF epidermal growth factor - TGF transforming growth factor     

Serum-dependent effects of IGF-I and insulin on proliferation and invasion of human first trimester trophoblast cell models

Extravillous cytotrophoblasts are specialised epithelial cells of the placenta that proliferate or invade the maternal decidua. Little is known about the mechanisms that regulate these processes. Here the effects of several insulin and insulin-like growth factor-I (IGF-I) doses, either singly or in synergy with serum, on human chorionic gonadotropin-beta (hCG-beta) secretion (RIA), proliferation (cell counting, cyclin B(1) levels) and invasion [Matrigel invasion assay, secretion of matrix metalloproteinases (MMP) 2 and 9] were investigated. The choriocarcinoma cell lines BeWo, JAR and JEG-3 served as models for first trimester human trophoblasts. Both growth factors altered hCG-beta secretion and proliferation dependent on the cell line. Insulin stimulated proliferation in JAR cells and, to a lesser extent, in JEG-3 cells, and when cultured in serum-free medium, BeWo was not affected. Invasion was not affected although proMMP-2 levels in culture medium were altered under some conditions. A strong synergistic effect with serum was noted. In the presence of serum both growth factors reduced proliferation and invasion in a similar fashion. Since the cell models differ by their degree of differentiation, the data demonstrate that the effects of insulin and IGF-I strongly depend on serum and the degree of differentiation. It can be speculated that IGF-I can take on tasks of insulin in the regulation of trophoblast functions under conditions of insulinopenia.