ICAM-2 confers a non-metastatic phenotype in neuroblastoma cells by interaction with α-actinin.

Progressive metastatic disease is a major cause of mortality for patients diagnosed with multiple types of solid tumors. One of the long-term goals of our laboratory is to identify  molecular interactions that regulate metastasis, as a basis for developing agents that inhibit this process. Toward this goal, we recently demonstrated that intercellular adhesion molecule-2 (ICAM-2) converted neuroblastoma (NB) cells from a metastatic to a non-metastatic phenotype, a previously unknown function for ICAM-2. Interestingly, ICAM-2 suppressed metastatic but not tumorigenic potential in preclinical models, supporting a novel mechanism of regulating metastasis. We hypothesized that the effects of ICAM-2 on NB cell phenotype depend on the interaction of ICAM-2 with the cytoskeletal linker protein α-actinin. The goal of the study presented here was to evaluate the impact of α-actinin binding to ICAM-2 on the phenotype of NB tumor cells. We used in silico approaches to examine the likelihood that the cytoplasmic domain of ICAM-2 binds directly to α-actinin. We then expressed variants of ICAM-2 with mutated α-actinin-binding domains, and compared the impact of ICAM-2 and each variant on NB cell adhesion, migration, anchorage-independent growth, co-precipitation with α-actinin and production of localized and disseminated tumors in vivo. The in vitro and in vivo characteristics of cells expressing ICAM-2 variants with modified α-actinin-binding domains differed from cells expressing ICAM-2 wild type (WT) and also from cells that expressed no detectable ICAM-2. Like the WT protein, ICAM-2 variants inhibited cell adhesion, migration and colony growth in vitro. However, unlike the WT protein, ICAM-2 variants did not completely suppress development of disseminated NB tumors in vivo. The data suggest the presence of α-actinin-dependent and α-actinin-independent mechanisms, and indicate that the interaction of ICAM-2 with α-actinin is critical to conferring an ICAM-2-mediated non-metastatic phenotype in NB cells.

DNA-based drug interactions of cisplatin.

The interactions of cisplatin with other anti-cancer agents on the DNA level have been studied extensively in pre-clinical experiments. In general, combination of cisplatin with an antimetabolite, taxane, or topoisomerase inhibitor, can result in a modulation of platinum pharmacology on the DNA, for example, enhanced retention of the platinum-DNA adducts. These interactions are mostly sequence and cell type dependent. In cell line models, antimetabolites can enhance the number of platinum-DNA adducts, probably by inhibition of DNA repair pathways. However, in clinical trials, the opposite effect has been observed, with a reduction of these adducts upon combined treatment. For the taxanes it has been shown that they can inhibit the formation of platinum-DNA adducts, whereas topoisomerase I inhibitors increase the number of adducts, resulting in strong synergistic cytotoxicity. For this last interaction a mechanistic model has recently been proposed, in which the topoisomerase I enzyme directly binds to the platinum-DNA adduct. Thereafter, the topoisomerase I inhibitor binds to this complex, which yields large stabilised lesions to the DNA that are probably difficult to repair. Ongoing studies will proceed to elucidate the exact mechanism underlying the interactions between cisplatin and other anti-neoplastic agents on the DNA level. Such increased understanding might help in designing new and more effective treatment regimens for cancer. In this paper, we review the pre-clinical and clinical studies investigating the observed interactions between cisplatin, the antimetabolites, taxanes, and topoisomerase inhibitors on the DNA level.

Circumvention of breast cancer resistance protein (BCRP)-mediated resistance to camptothecins in vitro using non-substrate drugs or the BCRP inhibitor GF120918.

This study was aimed at characterizing the role of BCRP/MXR/ABCP (BCRP) in resistance of the human ovarian tumor cell lines T8 and MX3 to camptothecins more extensively and investigating whether resistance can be reversed by inhibiting BCRP by GF120918. Camptothecins studied were topotecan, CPT-11, and its active metabolite SN-38, 9-aminocamptothecin, and the novel experimental camptothecins NX211, DX8951f, and BNP1350. Notably, DX8951f and BNP1350 appeared to be very poor substrates for BCRP, with much lower resistance factors observed both in T8 and MX3 cells than observed for the other camptothecins tested. In the presence of a nontoxic dose level of GF120918, the intracellular accumulation of topotecan in the T8 and MX3 cells was completely restored to the intracellular levels observed in the sensitive IGROV1 parental cell line. This resulted in almost complete reversal of drug resistance to topotecan and to most of the other topoisomerase I drugs tested in the T8 cell line and to complete reversal in the MX3 cells. However, coincubation of DX8951f or BNP1350 with GF120918 did not affect the cytotoxicity of either of these drugs significantly. From the combined data, we conclude that the affinities of topoisomerase I drugs for BCRP are, in decreasing order: SN-38 > topotecan > 9-aminocamptothecin approximately CPT-11 > NX211 > DX8951f > BNP1350. Furthermore, GF120918 appears to be a potent reversal agent of BCRP-mediated resistance to camptothecins, with almost complete reversal noted at 100 nM. Potential BCRP-mediated resistance to topoisomerase I inhibitors can also be avoided by using the BCRP-insensitive drugs DX8951f or BNP1350. This observation may have important clinical implications for future development of novel camptothecins.

32P-postlabeling assay for the quantification of the major platinum-DNA adducts.

To allow more sensitive, selective, and routine analyses of platinum(Pt)-GG and -AG intrastrand cross-links we have significantly improved our quantitative (32)P-postlabeling assay (M. J. P. Welters et al. Carcinogenesis 18, 1767-1774, 1997). Instead of off-line scintillation counting we introduced an on-line flow radioisotope detector into the HPLC system. Furthermore, the isolation protocol for the adducts was significantly modified and optimized to reduce interfering background peaks that prevented quantification of low levels of the cisplatin-DNA adducts in white blood cells obtained from patients. Reduction of background signals was obtained by boiling the samples, followed by phenol/chloroform/isoamylethanol extraction after the DNA digestion step. The labeling efficiency for the adducts was increased by 40% by using Na-formate instead of NH(4)-formate for elution of the adducts from the strong cation-exchange columns. Finally, a calibration curve and quality controls were implemented. The labeling efficiencies were not different between the dinucleotides. The between- and within-run precision for the Pt-GG and Pt-AG adducts measured at the lower limit of quantification of 87 and 53 amol/microg DNA, respectively, was less than 20% CV. The adducts were stable in DNA stored for a 2-month time period at -80 degrees C. The assay is now routinely used for high-precision analyses of patient and cell line samples containing very low adduct levels.

Overexpression of the BCRP/MXR/ABCP gene in a topotecan-selected ovarian tumor cell line.

Topotecan- or mitoxantrone-selected cell lines (T8 and MX3, respectively), derived from the human IGROV1 ovarian cancer cell line, were resistant to the topoisomerase I inhibitors topotecan, SN-38 (the active metabolite of irinotecan), and 9-aminocamptothecin, as well as to the topoisomerase II drug mitoxantrone. In both resistant cell lines, decreased accumulation of topotecan and mitoxantrone was observed, caused by enhanced energy-dependent efflux of the drugs involved. In both cell lines, we found that the breast cancer resistance protein/mitoxantrone resistance/placenta-specific ATP binding cassette (BCRP/MXR/ABCP) gene was overexpressed. Furthermore, BCRP/MXR/ABCP expression levels in various partially revertant T8 cells correlated with the levels of resistance to topotecan, SN-38, and mitoxantrone, strongly suggesting BCRP/MXR/ABCP to be the transporter responsible for the enhanced efflux. Pharmacodynamic analysis demonstrated that BCRP/MXR/ABCP is a very efficient transporter of topotecan; in vitro, 70% of the intracellular topotecan pool was transported out of the T8 or MX3 cells within 30 s. In conclusion, we report for the first time that BCRP/MXR/ABCP can also be up-regulated upon exposure of tumor cells to the clinically important drug topotecan, and that BCRP-mediated efflux of topotecan is very efficient. This highly efficient efflux of topotecan by BCRP/MXR/ABCP may have clinical relevance for patients being treated with topotecan.

Effects of c-myc oncogene modulation on differentiation of human small cell lung carcinoma cell lines.

Amplification and over-expression of oncogenes of the myc family are related to the prognosis of certain solid tumors such as small cell lung cancer (SCLC). For SCLC, c-myc is the oncogene most consistently found to correlate with the end stage behaviour of the tumour, in particular with survival after chemotherapeutic treatment. C-myc is important in many cellular processes such as proliferation, differentiation and apoptosis. In the present study the relationship between c-myc and differentiation was analyzed by down-regulation of endogenous c-myc protein, using two approaches: first by coculturing with antisense (AS) oligodeoxynucleotides (ODN) in the human SCLC cell line GLC4 and its 6-fold cisplatin resistant subline GLC4-CDDP, second by stable transfection of GLC4-CDDP with a dexamethasone-inducible AS c-myc expression vector. Basic characterization of the differentiation status of GLC4 and GLC4-CDDP showed a decrease in neuroendocrine differentiation in GLC4-CDDP compared to GLC4 Cytokeratin was absent in both cell lines. No significant differences in expression of adhesion molecules or myeloid antigens were observed between the lines. Vimentin expression was higher in GLC4-CDDP compared to GLC4 (AS c-myc ODN)-induced growth inhibition and down-regulation of endogenous c-myc protein further decreased neuroendocrine differentiation (CD57 positive cells) in GLC4-CDDP without affecting the expression of other antigens such as vimentin (intermediate filament),CD15 (myeloid antigen) and VLA-alpha 4 (adhesion molecule) and did not alter the expression of these antigens in GLC4 (AS c-myc RNA)-induced growth inhibition did not significantly affect the expression of the tested antigens in the AS c-myc transfected GLC4-CDDP/AS cell line. No effect of nonsense c-myc ODN or dexamethasone-induced control RNA (controls) was observed.

Effects of an inducible anti-sense c-myc gene transfer in a drug-resistant human small-cell-lung-carcinoma cell line.

Small-cell-lung-cancer (SCLC) is characterized by rapid development of resistance to cytotoxic agents, such as cisplatin (cDDP) and anthracyclines. c-myc over-expression is one of the reported genetic alterations in this tumor. Amplification of the c-myc gene in this and other cancers is often correlated with poor prognosis. We studied the existence of a correlation between resistance and activation of the c-myc oncogene in a cDDP-resistant SCLC sub-line, GLC4cDDP, containing a c-myc gene amplification. This cell line was stably transfected with either an anti-sense c-myc (AS) or control (C) expression vector resulting in the sub-clones GLC4cDDP/AS and GLC4cDDP/C respectively. PCR and RT-PCR analysis illustrated the integration and activation of the dexamethasone(dex)-inducible MMTV-LTR promoter linked to the complete AS-c-myc fragment in GLC4cDDP/AS cells, but not in GLC4cDDP/C cells. Dex-induced AS-c-myc RNA resulted in 50% growth inhibition during the first 48 hr, which declined to 25% at 72 hr. In addition, AS-c-myc RNA expression reduced the cloning efficacy by 36% and induced 2-fold more apoptosis within 24 hr in GLC4cDDP/AS cells. Dex treatment did not affect the proliferation, clonogenicity and induction of apoptosis in the control cell lines. Furthermore, AS-c-myc RNA expression caused a 1.4-fold increased cDDP sensitivity but no change in doxorubicin or vincristine sensitivity in GLC4cDDP/AS cells. Our results indicate that AS-c-myc RNA expression causes inhibition of cell proliferation, induces apoptosis, reduces clonogenicity and interferes with cDDP sensitivity but not with doxorubicin or vincristine sensitivity.

Effects of c-myc oncogene modulation on drug resistance in human small cell lung carcinoma cell lines.

Small cell lung carcinoma (SCLC) is characterized by rapid development of resistance to drugs, such as cisdiamminedichloroplatinum (II) (cDDP) and anthracyclines. The molecular basis for resistance to cDDP and adriamycin (Adr) is poorly understood. One of the genetic alterations observed in SCLC, which is correlated with poor prognosis, is amplification and overexpression of c-myc oncogene. Therefore, activation of the c-myc oncogene might form a basis for resistance. The relationship between c-myc and cDDP as well as Adr resistance was analyzed by down-regulation of endogenously expressed c-myc in the human SCLC cell line GLC4, and its cDDP and Adr resistant sublines (GLC4-cDDP and GLC4-Adr). Cells were incubated with an unmodified antisense (AS) oligodeoxynucleotide complementary to the first 5 codons of the c-myc mRNA in serum free culture medium. Pre-incubation with 15 microM AS c-myc, reduced c-myc protein expression and induced 30-35% growth inhibition in all 3 cell lines. It resulted in increased cDDP sensitivity in GLC4-cDDP but not in GLC4. However, this pre-incubation did not affect Adr sensitivity in all lines. The effect of AS c-myc pretreatment on cDDP resistance was not mediated by changes in cell cycle distribution. These findings suggest that c-myc plays a role in cDDP resistance by effects other than those on cell cycle distribution.