Effect of urine on clonal growth of human bladder cancer cell lines

Human urine contains growth factors; their physiological roles have not been established. The effect of normal human urine was examined in vitro on clonal growth of human bladder cancer cell lines. Clonal growth of HT-1376, HT-1197, and T24 was enhanced by five different fresh human urine samples from young men. Colony stimulating activity was detected in fractions with a molecular weight greater than 5000 by ultrafiltration. Sephadex G-50 gel chromatography identified two peaks of colony stimulative activity in HT-1376 with molecular weights of approximately 6000 and greater than 12,400, respectively; these two peaks also possessed immunoreactive epidermal growth factor (EGF) and NRK-49F transforming activities. The three bladder cancer cell lines possessed large quantities of EGF specific binding sites and exogenous EGF stimulated colony formation; EGF concentrations in human urine were found to be remarkably higher than those of exogenously added EGF which stimulated clonal growth of bladder cancer cell lines. Moreover, it was demonstrated that fresh urine samples (5%) incubated with anti-human EGF monoclonal antibody (KEM-10) neutralized completely the colony stimulating effects in HT-1376. These results indicate that fresh human urine stimulates clonal growth in human bladder cancer cell lines and that a major part of the activity is represented by urinary EGF. The data promote urinary EGF as a progressive agent of human bladder cancer.     

Receptor-targeted therapy of human experimental urinary bladder cancers with cytotoxic LH-RH analog AN-152 [AEZS- 108

Many bladder cancers progress to invasion with poor prognosis; new therapeutic methods are needed. We developed a cytotoxic LH-RH analog, AN-152 (AEZS-108) containing doxorubicin (DOX), for targeted therapy of cancers expressing LHRH receptors. We investigated the expression of LH-RH receptors in clinical bladder cancers and in HT-1376, J82, RT-4 and HT-1197 human bladder cancer lines. The effect of analog, AN-152, on growth of these tumor lines xenografted into nude mice was analyzed. Using molecular and functional assays, we also evaluated the differences between the effects of AN-152, and DOX alone. We demonstrated the expression of LH-RH receptors on 18 clinical bladder cancers by immunohistochemistry and on four human urinary bladder cancer lines HT-1376, J82, RT-4 and HT-1197 by Western blotting and binding assays. AN-152 powerfully inhibited growth of these bladder cancers in nude mice. AN-152 exerted greater effects than DOX and was less toxic. DOX activated strong multidrug resistance mechanisms in RT-4 and HT-1197 cancers, while AN-152 had no or less such effect. PCR assays and in vitro studies revealed differences in the action of AN-152 and DOX on the expression of genes involved in apoptosis. These results suggest that targeted cytotoxic LH-RH analog, AN-152 (AEZS- 108), should be examined for treatment of patients with LH-RH receptor positive invasive bladder cancers.     

Differential sensitivities of human testicular and bladder tumor cell lines to chemotherapeutic drugs

The in vitro drug sensitivities of 5 human testicular tumor cell lines (Tera II, SuSa, NEC-8, 833K, T3B1) and 5 human bladder carcinoma cell lines (RT4, RT112, T24, HT1197, HT1376) were compared. Cytotoxicities of cisplatin and doxorubicin were assessed by inhibition of colony-forming ability during continuous exposure to a range of drug concentrations. The ranges of the drug concentrations required to kill 70% of clonogenic cells obtained against the testicular cell lines were 1-7 ng/ml and 21-161 ng/ml for doxorubicin and cisplatin, respectively, compared with 4-19 ng/ml and 112-431 ng/ml for the bladder cell lines. This study shows that continuous cell lines retain the relative clinical chemosensitivities of their tumors of origin. The results also indicate that testicular tumor cells are inherently more sensitive to the cytotoxic effects of chemotherapeutic drugs than are bladder cancer cells.     

Chondroitin sulphate enhances the antitumor activity of gemcitabine and mitomycin-C in bladder cancer cells with different mechanisms

Non-muscle invasive bladder cancer is the most common type of bladder cancer in Western countries. The glycosaminoglycan (GAG) layer at the bladder surface non-specifically blocks the adherence of bacteria, ions and molecules to the bladder epithelium and bladder cancer cells express CD44 that binds GAG. Currently, there are few options other than cystectomy for the management of non-muscle invasive bladder cancer with intravesical chemotherapy using several drugs such as gemcitabine (GEM) and mitomycin-C (MMC) with poor prophylactic activity. In this study, we investigated the effects of the GAG chondroitin sulphate (CS) on the growth inhibition of human bladder cancer cell lines HT-1376 and effects of the combination between GEM or MMC with CS. We have found that CS, MMC and GEM induced 50% growth inhibition at 72?h. Therefore, we have evaluated the growth inhibition induced by different concentration of CS in combination with MMC or GEM, respectively, at 72?h. We have observed, at Calcusyn analysis, a synergism when HT-1376 cells were treated with CS in combination with MMC or GEM, respectively, if used at an equimolar ratio. We have also found that CS/GEM combination induced a strong potentiation of apoptosis with the consequent activation of caspases?9 and 3. On the other hand, HT-1376 cells were necrotic if exposed to the CS/MMC combination and no signs of caspase activation was observed. In conclusion, in the human bladder cancer cell line HT-1376 pharmacological combination of CS with GEM or MMC resulted in a strong synergism on cell growth inhibition.     

Elevated expression of DNA topoisomerase II in camptothecin-resistant human tumor cell lines

In a previous study, we established camptothecin (CPT)-resistant cell lines, A549/CPT and HT-29/CPT, from human lung cancer A549 and human colon cancer HT-29. A549/CPT was shown to express similar amounts of DNA topoisomerase I (topo I) as the parental line, and HT-29/CPT was shown to express lower amounts of topo I than its parental line. DNA topoisomerases I and II are known to be functionally related. In the present study, the possible alterations in topo II expression were examined in these human CPT-resistant lines. In A549/CPT and HT-29/CPT, the cellular contents of topo II and its mRNA were elevated over that seen in each parental line. Nuclear extracts from A549/CPT and HT-29/CPT showed higher topo II activity than those from the corresponding parental lines when the same amounts of nuclear protein were used. Topo II was partially purified from HT-29 and HT-29/CPT by hydroxylapatite column chromatography, and the enzyme activities were compared. HT-29/CPT showed higher topo II activity in the hydroxylapatite column-eluted fractions than HT-29. These results indicate the possible activation of topo II expression in the CPT-resistant cell lines.     

Mycobacterium phlei cell wall complex directly induces apoptosis in human bladder cancer cells

Intact mycobacteria and mycobacterial cell wall extracts have been shown to inhibit the growth of human and murine bladder cancer. Their mechanism of action is, however, poorly understood. Mycobacterium phlei mycobacterial cell complex (MCC) is a cell wall preparation that has mycobacterial DNA in the form of short oligonucleotides complexed on the cell wall surface. In this study, we have investigated the possibility that MCC has anti-cancer activity that is mediated by two different mechanisms – a direct effect on cancer cell proliferation and viability and an indirect effect mediated by the production of interleukin 12 (IL-12), a cytokine known to possess anti-cancer activity. We have found that, although MCC is a potent inducer of IL-12 and IL-6 synthesis in monocytes and macrophages either in vitro or in vivo, it is unable to induce the synthesis of either IL-12, IL-6 or granulocyte–macrophage colony-stimulating factor (GM-CSF) by the human transitional bladder cancer cell lines HT-1197 and HT-1376. MCC is not directly cytotoxic towards these cancer cells, but induces apoptosis as determined by nuclear DNA fragmentation and by the release of nuclear mitotic apparatus protein. Mycobacterium phlei DNA associated with MCC is responsible for the induction of apoptosis. Our results indicate that MCC directly effects bladder cancer cells by inhibiting cellular proliferation through the induction of apoptosis, and has the potential for an indirect anti-cancer activity by stimulating cancer-infiltrating monocytes/macrophages to synthesize IL-12. © 1999 Cancer Research Campaign     

Comparison of DNA interstrand cross-linking and strand breakage by 1,3-bis(2-chloroethyl)-1-nitrosourea in polyamine-depleted and control human adenocarcinoma cells

Recent evidence from our laboratory and from others suggested that pretreatment with alpha-difluoromethylornithine (DFMO) sensitizes some human and rodent tumor cell lines to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Many human tumor cells are resistant to chloroethylnitrosourea-induced DNA interstrand cross-linking and cell kill due to their high levels of the DNA repair protein O6-alkylguanine DNA alkyltransferase. We therefore investigated DFMO-mediated sensitization to BCNU in BCNU-sensitive and -resistant cells. Colony formation assays were used to compare BCNU cytotoxicity in DFMO-pretreated and control cultures of two colon tumor lines, HT-29 cells, which have high alkyltransferase levels and thus are BCNU-resistant, and BE cells, which are deficient in this repair capacity and thus are BCNU-sensitive. Polyamine depletion significantly enhanced BCNU cytotoxicity only for the repair-proficient HT-29 cell line. BE cells were 40-fold more sensitive to BCNU than were HT-29 cultures. However, in BE cells, no effect of polyamine depletion was found on cellular response to BCNU treatment at 72 h after DFMO treatment. Reverse-phase high-performance liquid chromatography assays of polyamine concentrations in cell extracts verified that DFMO produced comparable degrees of polyamine depletion for both cell lines. DNA alkaline elution analysis was used to monitor BCNU-induced formation of DNA single strand breaks, DNA interstrand cross-links, and DNA-protein cross-links. Equal concentrations of BCNU produced similar levels of strand breaks and DNA-protein cross-links in DFMO-pretreated and control cultures for both cell lines. These data suggest that DNA in polyamine-deficient HT-29 and BE cells is not more accessible to BCNU than is DNA in controls. No DNA interstrand cross-links were detected in either DFMO-pretreated or control HT-29 cells after BCNU treatment. Further, in BE cells which accumulate BCNU-induced DNA interstrand cross-links, no increase in the measureable levels of cross-links resulted from polyamine deficiency. Our observations suggest that mechanisms other than increased DNA interstrand cross-link formation may be mediating the enhanced efficacy of BCNU in polyamine-deficient HT-29 cell cultures. Our findings may also imply that cellular targets for BCNU other than DNA damage may be responsible for DFMO-induced chemosensitization in the repair-proficient cells.     

Cytocidal effect of purified human fibroblast interferon on tumor cells in vitro

RT-4 tumor cells, derived from human carcinoma of urinary bladder, were destroyed following exposure to partially purified human fibroblast interferon (IFN). The cytocidal effect of IFN was detected after addition of more than 200 IU IFN/ml and incubation of cell cultures for 2 days or longer. This effect was observed as morphologic changes and decrease in either dye uptake as morphologic changes and decrease in either dye uptake or colony formation by the IFN-treated cells. The cytocidal response of RT-4 tumor cells to IFN was the most pronounced, whereas the responses of three other tumor cell lines [HT-29 (colon adenocarcinoma) and SAOS-2 and 5959 (osteosarcoma)] were markedly weaker. Diploid fibroblasts were completely resistant to the cell-killing effect of IFN. Susceptibilities of the four tumor cell lines and two normal fibroblasts strains tested to the three effects of IFN (cytocidal, antiproliferative, and antiviral) appeared to be distinct.     

Cross-sensitivity to x-radiation and type-I and type-ii DNA topoisomerase inhibitors in a range of human and rodent cell-lines

We compared the relative X-radiation response of confluent (i.e. essentially non-proliferating) cultures of three human tumor (U-87MG, Mel-3, HT-144), one human normal (AG1522) and two rodent normal (AA8, V3) cell lines to their relative sensitivities to a DNA topoisomerase (topo) II poison (etoposide) and to a topo I poison (camptothecin). The relative sensitivity of these cell lines to etoposide (for 8 h exposure at 37 degrees C) is extremely similar to their relative X-radiation sensitivity, suggesting a direct correlation between X-radiation sensitivity and susceptibility to killing by topo II poison. The relative sensitivities of these cell lines to camptothecin (also 8 h, 37 degrees C exposure) also agree generally with their relative X-radiation sensitivities although the correlation is not as good as for etoposide. In addition, exponential phase (i.e, actively proliferating) cultures of the radiosensitive HT-144 cells are more susceptible to killing by both etoposide and camptothecin than the radioresistant Mel-3, confirming previously reported cross-sensitivities between X-radiation and topo poisons in actively proliferating cultures of other types of cell lines. Hence our results suggest that the previously reported cross-sensitivity between topo II poisons and X-radiation in actively proliferating rodent cell lines is also observed in 'non-proliferating' rodent and human cell lines. Additionally, there is cross-sensitivity between topo I poisons and X-radiation in both rodent and human cell lines as well.     

Human bladder carcinoma cell lines as indicators of oncogenic change relevant to urothelial neoplastic progression.

Analysis of human tumour-derived cell lines has previously resulted in the identification of novel transformation-related elements and provided a useful tool for functional studies of different genes. To establish the utility of such cell lines as indicators of change relevant to urothelial cancer, we have characterised the expression of five genes (p53, MDM2, Rb, E-cadherin, APC) within a panel of human bladder carcinoma cell lines. Using single-strand conformation polymorphism (SSCP) and direct sequencing, p53 mutations were identified in 7/15 (47%) cell lines reflecting events reported in bladder tumours. Immunohistochemical analysis of p53 in cultured cells and in paraffin-embedded sections of xenografts from the cell line panel revealed discordant results. An absence of p53 nuclear staining was associated with an exon 5 mutation in EJ and with multiple p53 mutations found in J82. Two cell lines positive for p53 staining in the absence of detectable mutation displayed overexpression of MDM2 (PSI, HT1197) in Western blot analysis. Loss or aberrant Rb expression was recorded in 5/15 (TCCSUP, SCaBER, 5637, HT1376, J82) cell lines. Absence of E-cadherin was recorded in 5/15 cell lines (TCCSUP, EJ, KK47, UM-UC-3, J82) with loss of alpha-catenin in immunoprecipitated E-cadherin complexes of CUBIII. Western blot analysis of APC revealed a truncated protein in 1/15 (CUBIII) cell lines. The characterisation of oncogenic events within this panel of human bladder carcinoma cell lines establishes a representation of change observed in bladder tumours and better defines the genotypic background in these experimental human cell models of neoplastic progression.     

The invasive features of glial and non-central nervous system tumor cells are different on organotypic brain slices from newborn rats

On radiograms, glial tumors are usually seen to invade in a finger-like fashion, while non-central nervous system (CNS) tumors expand in a mass-like fashion. We prepared organotypic brain slice cultures from newborn rats to investigate the invasive behavior of human brain tumors using glial tumor cell lines (U-87MG, U-373MG, U-251MG, and SF-126) and of non-CNS tumors using cell lines; HT-1080 (human malignant fibrosarcoma), RFRF (human lung carcinoma), MIA-PICA (human pancreatic carcinoma), and Colo38 (human malignant melanoma). We selected an area that was centered at a junction between deep cortical layers and the striatum, punched a hole measuring 0.5-0.7 mm in diameter in this area, implanted different rhodamine-labeled tumor cells at that site, and observed their invasive behavior under an inverted fluorescent microscope. Over the course of several days, all glioma cells invaded in a finger-like fashion; U-373 MG cells invaded farthest. Non-CNS tumors expanded in mass-like fashion into adjacent areas. Using the slice cultures, we also investigated the regulatory effect on tumor invasion of forced expression of glial fibrillary acidic protein (GFAP) after gene transfection. The forced expression of GFAP rendered U-87MG and HT-1080 cells less invasive. Our results show that organotypic brain slice cultures are an excellent medium for studying the invasive features of glial and non-CNS tumors.     

Comparison of the growth and metastasis of four human intestinal tumor cell line xenografts

The growth and metastasis of four human intestinal tumor cell lines: one duodenal adenocarcinoma (HTB-40) and three adenocarcinomas of the colon (CCL-218, CCL-222 and HT-29) have been compared in vitro and in vivo in nude mice. HTB-40 was the fastest growing cell line in vitro with a doubling time (DT) of 14.8 h. CCL-218 and CCL-222 grew more slowly in vitro with doubling times of 21.6 and 22.8 hours, respectively. All three of these tumors grew more slowly in vivo with doubling times ranging from 39.1 h (CCL-218 in male nude mice) to 65.3 h (CCL-222). The growth of CCL-218 cells was significantly slower in female nude mice DT 51.0 h). HT-29 was the slowest growing in vitro (DT 23.8 h) and in vivo (DT about 100 h). HT-29 also showed the greatest discrepancy between its DT measured in vivo as compared to in vitro, suggesting a greater clonogenic cell loss from HT-29 tumors in vivo. Histologic evaluation of these tumors grown subcutaneously in nude mice showed all to be anaplastic and to produce liver micrometastases. However, more extensive abdominal and liver metastases were observed in the nude mice injected with HT-29 cells, and some of these metastases had morphologic features of moderately well-differentiated epithelium. These results indicate the usefulness of the HT-29 tumor cell line as an experimental model of metastasis from a human colonic adenocarcinoma.     

Multicellular tumor spheroids in serum-free culture

The serum-free culture of multicellular tumor spheroids from two rat (BT4c, BT5c) and two human (GaMg, D-54Mg) glioma cell lines is described. The spheroids were propagated in two different chemically defined media, showing differences in growth requirements between the human and rat cell lines. The spheroids from all the cell lines showed reduced growth rate as compared to spheroids maintained in serum supplemented culture. A change in spheroid morphology was observed for the D-54Mg cell line, indicated by a reduced occurrence of microvilli, increased pyknosis and cellular granularity. The other spheroids maintained their morphology in serum-free culture. For the D-54 Mg spheroids, flow cytometric DNA measurements showed that the serum-free growth conditions caused a drop in the DNA S-phase (6.4% vs. 12.3% in serum supplemented cultures). The cell cycle distribution was unchanged for the other cell lines tested. The present study provides the basis for using multicellular tumor spheroids in biological studies which are dependent on a detailed control with chemical environment.     

Shared antigens between BCG and tumor cells--immunotherapy with BCG for mouse tumor

Cytotoxic activity of anti-BCG rabbit serum (A-BCG) and the antigenic relationship between BCG and tumor cells were studies. A-BCG showed high cytotoxic activity against line 10 tumor cells of guinea pig Strain 2 and Colon 26 tumor cells of BALB/c mouse but not to cells of human bladder cancer (HT 1197, HT 1376). Cell-killing activity of A-BCG against line 10 and Colon 26 was dependent on complement participation. It was also found that Colon 26 cells were heterogenous in their susceptibility to the killing activity of A-BCG. Antigenic studies between BCG and tumor cells were undertaken using on indirect immunofluorescence method and it was found that all tumor cells used in the experiment shared common antigens with BCG. In immunotherapy with BCG for Colon 26 tumors, BCG worked effectively for suppressing tumor growth. However, enhancement of tumor growth in some of the BCG-treated mice was observed.     

Sucrase-isomaltase: a marker of foetal and malignant epithelial cells of the human colon

The presence of sucrase-isomaltase (SI), a glycoprotein hydrolase normally restricted to the brush border membrane of the enterocytes of the small intestine, was investigated in tumours which developed in nude mice inoculated with six human colon carcinoma cell lines (HT-29, Caco-2, HRT-18, HCT-8R, SW-480, and CO-115). Foetal and normal adult human small intestines and colons were used as controls. SI was studied by (1) immunofluorescence with rabbit antibodies raised against purified human small intestine SI; (2) polyacrylamide gel electrophoresis and immunoblotting; and (3) determination of the enzyme activity. SI was antigenically present, and enzymatically active, in all the tumours derived from Caco-2 and HT-29 cells. The presence of the enzyme was associated with that of typical brush borders at transmission electron microscopy examination. SI was absent from the tumours developed with the other four cell lines, as well as from the normal adult colon mucosa. SI was also present and active in the colons of mid-gestation foetuses, ranging in ages between 20 and 28 weeks; it was absent from the colons of late-gestation foetuses. The presence of SI in tumours derived from two cell lines suggests that this enzyme is a marker, so far unsuspected, of certain human colon cancers, and that the differentiation pattern of these particular cancers closely resembles that of the foetal colon.     

Determinants of the cytotoxicity of irinotecan in two human colorectal tumor cell lines

PURPOSE: Irinotecan is a drug of the camptothecin family that has proven activity in advanced colon cancer, with about 20% responses in untreated as well as in 5-fluorouracil-resistant tumors. Irinotecan is considered as a prodrug which needs to be activated to SN-38 by carboxylesterases to become able to interact with its target, topoisomerase I. The work reported here intended to identify the determinants of the cytotoxicity of irinotecan in two human colorectal tumor cell lines, LoVo and HT-29, at the level of the target of the drug and at the level of the availability of the active metabolite to the target. RESULTS: The cytotoxicity of irinotecan and SN-38 markedly differed in the two cell lines: irinotecan IC(50) values were 15.8 microM for LoVo cells and 5.17 microM for HT-29 cells; SN-38 IC(50) values were 8.25 n M for LoVo cells and 4.50 n M for HT-29 cells. Topoisomerase I expression (at the mRNA and the protein levels) and catalytic activity were similar in the two cell lines. Irinotecan induced similar amounts of cleavable complexes at its IC(50) in both cell lines. SN-38 induced a concentration-dependent formation of cleavable complexes, which was not significantly different in the two cell lines. Expression of the carboxylesterase CES1 was higher in HT-29 than in LoVo cells. Expression of the carboxylesterase gene CES2 was comparable in the two cell lines and much higher than CES1 gene expression. Carboxylesterase activity was extremely low using p-nitrophenylacetate as a substrate (1.45 and 1.84 pmol/min per mg proteins) and could not even be detected using irinotecan as a substrate. Cell accumulation of irinotecan was markedly different, reaching consistently higher levels in HT-29 cells than in LoVo cells. CONCLUSIONS: Our results indicate that (1) the cytotoxicity of irinotecan was likely due to the drug itself and not to its metabolite SN-38, and (2) that irinotecan uptake was more predictive of its cytotoxicity than topoisomerase I availability and activity in these two cell lines.     

Effect of incadronate on proliferation of mesenchymal tumor cells with or without activated Ras mutation

The present investigation examined the effect of bisphosphonates on six mesenchymal tumor cell lines and the mechanisms of inhibition of tumor cell proliferation. HT-1080, a fibrosarcoma cell line that exhibits increased Ras activity due to a mutation of the Ras gene, demonstrated significantly reduced tumor cell proliferation upon treatment with incadronate. The other cell lines, however, which lack mutation of the Ras gene, showed no influence upon treatment with incadronate. Autoradiography demonstrated no difference in the uptake of 3H-labelled incadronate between susceptible and unaffected cells. The anti-proliferation of HT-1080 was reversed by the addition of geranylgeranyl pyrophosphate. Etidronate exhibited no influence on all tested cell lines. On the basis of these data, we hypothesized that incadronate inhibits the mevalonate pathway and blocks oncogenic Ras signaling. In an effort to confirm this hypothesis, the influence of incadronate on an oncogenic Ras transfected BALB/3T3 cell line (Bhas 42) and a parental BALB/3T3 cell line were compared. The parental BALB/3T3 cells showed slight inhibition upon treatment with incadronate, however, the proliferation of Bhas 42 cells was significantly reduced. These results suggest that incadronate suppresses oncogenic Ras-activated mesenchymal tumors through the inhibition of Ras signaling pathways.     

Insulin receptor regulation in cultured human tumor cells

Insulin binding to monolayer cell cultures of human fibroblasts, human colon carcinoma (HCT-8, HT-29), human breast carcinoma (MCF-7, T-47D), and melanoma (MM-96) was measured using 125I-insulin. Binding was time and temperature dependent in all cell lines, and only one cell line (MM-96) degraded 125I-insulin. High-affinity insulin-binding sites (Kd = 1.4 X 10(-9) M to 0.4 X 10(-10) M) were detected in all cell lines, and insulin-binding capacity ranged from 0.6 to 14 fmol/10(6) cells. Receptor down-regulation was studied by exposing cells to increasing concentrations of unlabeled insulin, dissociating surface-bound insulin and measuring residual receptors by 125I-insulin uptake. Exposure of tumor cells to greater than 10(-6) M insulin for 2 hr at 37 degrees led to a decrease in the number of insulin binding sites in MM-96 and colon cell lines only, with maximum down-regulation ranging from 58% (MM-96) to 88% (HCT-8) receptor loss. The decrease in insulin binding was due to a decreased number of receptors per cell with no change in affinity. Monolayers exposed to 1.7 X 10(-5) M unlabeled insulin for 7 hr at 37 degrees invariably showed greater than 50% receptor loss. However, monolayers exposed to 1.7 X 10(-8) M unlabeled insulin for 7 hr at 37 degrees showed less marked (0 to 39%) down-regulation. In comparison, human fibroblasts showed 57% receptor loss after exposure to 3.5 X 10(-9) M unlabeled insulin for 7 hr. Thus, markedly supraphysiological concentrations of insulin are required to down-regulate insulin receptors in tumor cell lines compared with normal cells. This suggests a tumor-associated resistance to receptor down-regulation.     

阿司匹林对人结肠癌细胞株SW480、HT29中VEGF、β-catenin表达的影响

目的 探讨阿司匹林对人结肠癌细胞株HT-29、SW480生长增殖的影响及其β-catenin、VEGF蛋白和mRNA的表达,并为结直肠癌的预防和治疗提供新的实验和理论依据。方法 采用MTT法检测阿司匹林对SW480、HT-29细胞生长增殖的影响;Real-time PCR和Western blot法检测HT-29、SW480细胞中VEGF、β-catenin mRNA及蛋白的表达情况。结果 在SW480、HT-29细胞的生长增殖过程中,阿司匹林对其有着显著的抑制作用,且具有量效、时效关系;除6 h干预组外,余各组阿司匹林均可抑制VEGF、β-catenin蛋白和mRNA的表达（P＜0.05）。结论 阿司匹林对人结直肠癌细胞株SW480、HT-29的生长增殖具有显著的抑制作用,并对SW480、HT-29中VEGF、β-catenin mRNA和蛋白质的表达具有显著的下调效应;而阿司匹林通过抑制β-catenin的表达,同时调节VEGF抑制肿瘤血管的生成可能是其预防和治疗结直肠癌的作用机制之一。     

Decreased expression of DNA topoisomerase I in camptothecin-resistant tumor cell lines as determined by a monoclonal antibody

DNA topoisomerase I (topo I) has been identified as a principal target of a plant alkaloid camptothecin (CPT) and its derivative (CPT-11). The latter compound is expected to be a clinically useful antitumor agent. Three human tumor cell lines resistant to CPT (A549/CPT, HT-29/CPT, St-4/CPT) were isolated in vitro, and a murine tumor cell line resistant to CPT-11 (P388/CPT) was isolated in vivo by continuous exposure of the drugs. A549/CPT, HT-29/CPT, and St-4/CPT showed 1.8-, 6.9-, and 8.8-fold more resistance to CPT, and P388/CPT showed 45-fold more resistance to CPT than did the parental line. To examine the possible involvement of topo I in drug-resistant mechanisms, a monoclonal antibody was developed by using purified human topo I as antigen. The antibody T14C (immunoglobulin G1) recognized both human and murine topo I, as shown by Western blot analysis. By using this monoclonal antibody, cellular contents of topo I were examined in CPT-resistant tumor lines. Respective contents of topo I in HT-29/CPT, St-4/CPT, and P388/CPT were approximately 8-, 4-, and 3-fold less than those in their parental cell lines. A549/CPT, a weak CPT-resistant line, possessed amounts of topo I similar to those of the parental line. HT-29/CPT showed lower topo I activity than did the parental HT-29 in the nuclear extracts and in the hydroxylapatite column-eluted fractions. Purified topo I from HT-29 and HT-29/CPT showed similar catalytic activity when the same amounts of protein were used. These results indicate that the quantitative reduction of topo I content seems to be the most frequently occurring event in the development of resistance to camptothecin.