Novel_target.com.
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Antineoplastic Agents↗
Biomedical subjects
T Fojo
Publications and source records attributed to T Fojo.
Wild-type p53 marginally induces endogenous MDR-1 mRNA without causing a measurable drug resistance in human cancer cells.
The notion that wt p53 downregulates MDR-1 links p53 mutations to multidrug resistant phenotype. Alternatively, it has been envisioned that wt p53 protects cells against DNA damaging drugs by inducing MDR-1. Opposing conclusions on the relationship between MDR-1 and p53 have been predominantly based on the effects of p53 on MDR-1 promoter-constructs. We found that introduction of wt p53 slightly induced MDR-1 mRNA in three cell lines having endogenous mt p53. Wt p53-mediated induction of endogenous MDR-1 may represent a rudiment of cellular protection against toxic compounds earlier in evolution. Marked induction of p21WAF1/CIP1 (p21) mRNA was observed in all cell lines; and lower levels of wt p53 were required to induce p21 than MDR-1. Pgp was undetectable and wt p53 did not increase resistance to an MDR-1 substrate, suggesting the changes in MDR-1 mRNA may be functionally insignificant. Unlike endogenous MDR-1, the expression of an MDR-1 promoter (-434/+147 fragment) - luciferase construct was unchanged or even inhibited by wt p53 that may be secondary to wt p53-mediated cytotoxicity. Thus, partial promoter constructs may not accurately represent endogenous MDR-1.
Adenoviridae↗
The P-glycoprotein antagonist PSC 833 increases the plasma concentrations of 6alpha-hydroxypaclitaxel, a major metabolite of paclitaxel.
PURPOSE: Overexpression of P-glycoprotein (Pgp) is one mechanism of drug resistance in cancer chemotherapy. A Phase I trial was conducted using PSC 833, a Pgp antagonist, in combination with paclitaxel in patients with refractory cancer. The objective of this study was to assess the effect of PSC 833 on the metabolism of paclitaxel and characterize the differences in 6alpha-hydroxypaclitaxel pharmacokinetics. In addition, we examined the possibility of enhanced cytotoxicity of paclitaxel by the coexistence of 6alpha-hydroxypaclitaxel. EXPERIMENTAL DESIGN: Patients received paclitaxel 35 mg/m(2)/day by continuous intravenous infusion (CIVI) x 4 days without PSC 833 in cycle 1 and escalating doses of paclitaxel (13.1, 17.5, or 21.3 mg/m(2)/day CIVI x 4 days) with 5 mg/kg PSC 833 by mouth every 6 h x 7 days in cycle 2. Plasma samples were analyzed for both paclitaxel and its major metabolite with high-performance liquid chromatography methods. Using human liver microsomes, we studied the effect of PSC 833 on the metabolism of paclitaxel. In addition, the in vitro cytotoxicity of 6alpha-hydroxypaclitaxel alone and in combination with paclitaxel was evaluated. RESULTS: Twenty-one of 22 patients had a metabolite peak (6alpha-hydroxypaclitaxel) observed in the chromatogram of plasma samples from cycle 2 when they received paclitaxel in combination with PSC 833. This metabolite was not detectable in plasma obtained during the first cycle when they received paclitaxel without PSC 833. During cycle 2, the mean concentrations of 6alpha-hydroxypaclitaxel and paclitaxel were 0.10 +/- 0.074 and 0.079 +/- 0.041 microg/ml, respectively. A moderate association was observed between total bilirubin and 6alpha-hydroxypaclitaxel concentrations (P = 0.015, r = 0.52; n = 21). Human liver microsome experiments showed that a PSC 833 concentration as high as 10 microM did not affect the production of 6alpha-hydroxypaclitaxel. Paclitaxel cytotoxicity in HL60 and K562 human leukemia cells was increased in the presence of noncytotoxic concentrations of 6alpha-hydroxypaclitaxel. CONCLUSIONS: PSC 833 increases the plasma concentration of 6alpha-hydroxypaclitaxel during paclitaxel therapy. Inhibition of cytochrome P-450 3A4 by PSC 833 may explain this in part, although other mechanisms cannot be excluded.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Expression of beta-tubulin isotypes in human ovarian carcinoma xenografts and in a sub-panel of human cancer cell lines from the NCI-Anticancer Drug Screen: correlation with sensitivity to microtubule active agents.
Paclitaxel resistance has been associated with overexpression of P-glycoprotein and alterations involving tubulin. To investigate the clinical relevance of these in vitro resistance mechanisms, we established 12 human ovarian carcinoma xenografts, using samples from patients before the start of therapy or after paclitaxel treatment. These xenografts showed a wide range of sensitivity to paclitaxel, and in 4 of them, very low levels of multidrug resistance-1 expression were detected. Using quantitative PCR and human specific primers, the expression of five beta-tubulin isotypes was determined. HM40 was the predominant, accounting for 84.7-98.7% of all tubulin; expression of the other four isotypes (Hbeta9, Hbeta4, H5beta, and Hbeta2) was also detected but at lower levels. No correlation could be demonstrated between isotype expression and paclitaxel sensitivity in these 12 xenografts. A similar pattern of beta-tubulin isotype expression was observed in a subset of cell lines from the National Cancer Institute-Anticancer Drug Screen. In these cell lines, however, a significant correlation between increased expression of Hbeta4 isotype and resistance to paclitaxel was found. Taken together, these results suggest that altered expression of specific beta-tubulin isotypes may not play a significant role in paclitaxel sensitivity in vivo and argue against a possible significance in a clinical setting.
Animals↗
Altered topoisomerase IIalpha and multidrug resistance-associated protein levels during drug selection: adaptations to increasing drug pressure.
To understand resistance to topoisomerase II inhibitors, we used four cancer cell lines (ZR-75B, MDA-MB-231, T47D, and MCF-7) and performed a single-step selection process to isolate 50 clones resistant to topoisomerase II inhibitors. Of these, 26 were isolated with VP-16 and 24 with mAMSA. Sixteen of these isolates (four from each cell line; two selected with VP-16 and two with mAMSA) were further exposed to higher drug concentrations. Characterization of the resistant sublines revealed the adaptation that occurs with increasing drug concentration during in-vitro selections. Reduced topoisomerase IIalpha mRNA level was observed in the majority of the initial isolates. This reduction was accompanied by a decrease in topoisomerase II activity. Other isolates showed increased levels of multidrug resistance-associated protein (MRP). With advancing resistance, MRP expression was increased further, concomitantly with some recovery in topoisomerase IIalpha expression and topoisomerase II activity. In these sublines, high levels of resistance were attained as a result of synergism between the reduced topoisomerase IIalpha levels and MRP overexpression. These results extend previous studies demonstrating how cellular adaptation to increasing drug pressure utilizes more than one mechanism. Reduced expression of topoisomerase IIalpha occurs early in the selection process. MRP overexpression can occur early or can help to confer high levels of resistance. In the latter case, MRP overexpression allows some recovery of topoisomerase II activity without loss of high drug resistance.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Radiofrequency ablation of metastatic lesions in adrenocortical cancer.
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Adrenal Cortex↗
Loss of cell cycle control allows selective microtubule-active drug-induced Bcl-2 phosphorylation and cytotoxicity in autonomous cancer cells.
Lack of selectivity in the killing of tumor and normal cells is a major obstacle in cancer therapy. By inhibiting normal but not autonomous cell growth, we exploited the differences in cell cycle regulation to achieve a selective protection of nonautonomous cells against paclitaxel and other microtubule-active drugs. Tubulin polymerization, a primary effect of paclitaxel, can be dissociated from Bcl-2 phosphorylation and cytotoxicity in HL-60 cells. Growth arrest prevented paclitaxel-induced Bcl-2 phosphorylation and apoptosis without affecting paclitaxel-induced tubulin polymerization. We abrogated the effects of paclitaxel on MCF-10A immortalized breast cells, while preserving its effects on MCF-7 cancer cells. Unlike MCF-7 cells, MCF-10A cells were arrested by epidermal growth factor withdrawal, precluding paclitaxel-induced Bcl-2 phosphorylation. Furthermore, the inhibition of the epidermal growth factor receptor kinase with low doses of AG1478 arrested growth of MCF-10A but not MCF-7 cells. Pretreatment with AG1478 did not affect paclitaxel-induced Bcl-2/Raf-1 phosphorylation in MCF-7 but abrogated such phosphorylation in MCF-10A. Exploitation of growth factor dependency may allow the protection of normal cells from microtubule-active drugs.
Apoptosis↗
Paclitaxel selects for mutant or pseudo-null p53 in drug resistance associated with tubulin mutations in human cancer.
The efficacy of anticancer therapy is limited by the development of drug resistance. While the role of p53 in the intrinsic sensitivity of human cancer cells to paclitaxel (PTX) remains controversial, its role in acquired paclitaxel resistance has never been addressed. In this study we examined the p53 status of three paclitaxel selected human ovarian carcinoma sublines, resistant to paclitaxel due to acquired beta-tubulin mutations which impair paclitaxel's interaction with tubulin. In contrast to parental cells which have wt p53, in all PTX-resistant sublines p53 was functionally inactive. Two of the resistant sublines expressed high levels of transcriptionally inactive p53 protein, each with a distinct point mutation in codons 236 and 239 of the DNA binding domain. The third subline presented a novel p53 pseudo-null phenotype as a result of markedly decreased wt p53 mRNA expression. Introduction of ectopic wt p53 had no effect on PTX sensitivity in both parental and resistant cells, while it induced p21WAF1/CIP1, demonstrating an intact p53 pathway. While PTX resistance is primarily conferred by the tubulin mutations, the loss of functional p53 observed in all clones, suggests that this loss may facilitate the development of resistance potentially by providing a clonal advantage which promotes the isolation of paclitaxel resistant cells.
Antineoplastic Agents, Phytogenic↗
Alu-associated interstitial deletions and chromosomal re-arrangement in 2 human multidrug-resistant cell lines.
Previous studies have shown that gene re-arrangements play a significant role in tumorigenesis. Gene re-arrangements involving the human multidrug resistance-1 (MDR1) gene have been identified as a mechanism for MDR1 over-expression in human malignant cells. In 2 multidrug-resistant human cancer sublines with high levels of MDR1 and P-glycoprotein (MCF7/TX400 and S48-3s/Adr10), hybrid mRNAs containing sequences from MDR1 and an unrelated gene have previously been identified. To characterize and determine the site of the re-arrangements resulting in generation of hybrid mRNAs, we first constructed a lambda phage library extending over a contiguous genomic region of 100 kb and containing the region upstream of MDR1. In MCF7/TX400 cells, homologous recombination was observed involving an Alu repeat 80 kb upstream of the MDR1 gene, with a 79 bp intra-Alu deletion flanked by chi-like sequences at the re-arrangement junction. By contrast, non-homologous recombination was observed in S48-3s/Adr10 cells with Alu repeats near the junction sequence. While the specific features of the breakpoints appear to be different, Alu repeats might be involved in both gene re-arrangements. The gene re-arrangements at or near the Alu sequence should be regarded as potentially involved in the transcriptional activation of human MDR1.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Transactivation of the metallothionein promoter in cisplatin-resistant cancer cells: a specific gene therapy strategy.
BACKGROUND: Cisplatin (cis-diamminedichloroplatinum) is one of the most active agents against a broad range of malignancies, including ovarian cancer. Cisplatin resistance appears to be associated with several molecular alterations, including overexpression of metallothionein, a metal-binding protein. In the present study, we attempted to take advantage of metallothionein overexpression to overcome cisplatin resistance. METHODS: Using a virus-free system (liposomes), we sought to express the suicide gene, thymidine kinase (TK), driven by the promoter of the human metallothionein IIa (hMTIIa) gene using the pMT-TK plasmid. We used cisplatin-resistant human ovarian carcinoma cells as a model. RESULTS: We first analyzed metallothionein expression using a ribonuclease protection assay. In comparison to parental cells, the cisplatin-resistant cells were found to have increased expression of metallothionein messenger RNA (mRNA). Metallothionein overexpression in these cells was not associated with an increased copy number of the hMTIIa gene or with different transfection efficiencies. Furthermore, we showed by reverse transcription-polymerase chain reaction analysis that transfection of the pMT-TK plasmid results in a 56-fold higher expression of thymidine kinase mRNA in cisplatin-resistant cells compared with parental cells, consistent with increased metallothionein promoter-mediated transactivation in the cisplatin-resistant cells. Transfection of resistant cells with pMT-TK or a control plasmid (pCD3-TK) resulted in a marked sensitization to ganciclovir, with a 50% cell growth-inhibitory concentration (IC(50)) of 20 microg/mL and 9 microg/mL, respectively. Transfections of the cisplatin-sensitive cells resulted in no sensitization to ganciclovir with pMT-TK (IC(50) 200 microg/mL) and a high sensitization with pCD3-TK (IC(50) = 6 microg/mL). CONCLUSION: These studies suggest that pMT-TK gene therapy may provide an alternative treatment for cisplatin-refractory ovarian tumors.
Cisplatin↗
A common pharmacophore for epothilone and taxanes: molecular basis for drug resistance conferred by tubulin mutations in human cancer cells.
The epothilones are naturally occurring antimitotic drugs that share with the taxanes a similar mechanism of action without apparent structural similarity. Although photoaffinity labeling and electron crystallographic studies have identified the taxane-binding site on beta-tubulin, similar data are not available for epothilones. To identify tubulin residues important for epothilone binding, we have isolated two epothilone-resistant human ovarian carcinoma sublines derived in a single-step selection with epothilone A or B. These epothilone-resistant sublines exhibit impaired epothilone- and taxane-driven tubulin polymerization caused by acquired beta-tubulin mutations (beta274(Thr-->Ile) and beta282(Arg-->Gln)) located in the atomic model of alphabeta-tubulin near the taxane-binding site. Using molecular modeling, we investigated the conformational behavior of epothilone, which led to the identification of a common pharmacophore shared by taxanes and epothilones. Although two binding modes for the epothilones were predicted, one mode was identified as the preferred epothilone conformation as indicated by the activity of a potent pyridine-epothilone analogue. In addition, the structure-activity relationships of multiple taxanes and epothilones in the tubulin mutant cells can be fully explained by the model presented here, verifying its predictive value. Finally, these pharmacophore and activity data from mutant cells were used to model the tubulin binding of sarcodictyins, a distinct class of microtubule stabilizers, which in contrast to taxanes and the epothilones interact preferentially with the mutant tubulins. The unification of taxane, epothilone, and sarcodictyin chemistries in a single pharmacophore provides a framework to study drug-tubulin interactions that should assist in the rational design of agents targeting tubulin.
Alkaloids↗
Tubulin/microtubules: still a promising target for new chemotherapeutic agents.
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Antineoplastic Agents↗
p53 is associated with cellular microtubules and is transported to the nucleus by dynein.
Here we show that p53 protein is physically associated with tubulin in vivo and in vitro, and that it localizes to cellular microtubules. Treatment with vincristine or paclitaxel before DNA-damage or before leptomycin B treatment reduces nuclear accumulation of p53 and expression of mdm2 and p21. Overexpression of dynamitin or microinjection of anti-dynein antibody before DNA damage abrogates nuclear accumulation of p53. Our results indicate that transport of p53 along microtubules is dynein-dependent. The first 25 amino acids of p53 contain the residues that are essential for binding to microtubules. We propose that functional microtubules and the dynein motor protein participate in transport of p53 and facilitate its accumulation in the nucleus after DNA damage.
Active Transport, Cell Nucleus↗
Pretreatment with DNA-damaging agents permits selective killing of checkpoint-deficient cells by microtubule-active drugs.
Cell-cycle checkpoint mechanisms, including the p53- and p21-dependent G(2) arrest that follows DNA damage, are often lost during tumorigenesis. We have exploited the ability of DNA-damaging drugs to elicit this checkpoint, and we show here that such treatment allows microtubule drugs, which cause cell death secondary to mitotic arrest, to kill checkpoint-deficient tumor cells while sparing checkpoint-competent cells. Low doses of the DNA-damaging drug doxorubicin cause predominantly G(2) arrest without killing HCT116 cells that harbor wt p53. Doxorubicin treatment prevented mitotic arrest, Bcl-2 phosphorylation, and cell death caused by paclitaxel, epothilones, and vinblastine. In contrast, doxorubicin enhanced cytotoxicity of FR901228, an agent that does not affect microtubules. Low doses of doxorubicin did not arrest p21-deficient clones of HCT116 cells and did not protect these cells from cytotoxicity caused by microtubule drugs, but cells in which p21 expression was restored enjoyed partial protection under these conditions. Moreover, in p53-deficient clones of HCT116 cells doxorubicin did not induce either p53 or p21 and provided no protection against paclitaxel-induced cytotoxicity. Therefore, (a) p53-dependent p21 induction caused by doxorubicin protects from microtubule drug-induced cytotoxicity, and (b) pretreatment with cytostatic doses of DNA-damaging drugs before treatment with microtubule drugs results in selective cytotoxicity to cancer cells with defective p53/p21-dependent checkpoint.
Antineoplastic Agents↗
Construction of gene therapy vectors targeting adrenocortical cells: enhancement of activity and specificity with agents modulating the cyclic adenosine 3',5'-monophosphate pathway.
In preliminary studies we demonstrated that the CYP11B1 (11beta-hydroxylase) promoter could direct specific expression of a suicide gene in adrenocortical cancer cells, providing a potentially specific therapeutic option for adrenocortical cancer. In this present study we describe our attempts to enhance the activity of the CYP11B1 promoter while maintaining its specificity for adrenal cells. Using a putative enhancer element from the cholesterol side-chain cleavage (P450scc) gene, the activity of the CYP11B1 promoter in and its specificity for adrenocortical cells were enhanced. Treatment with 8-bromo-cAMP or forskolin resulted in further enhancement. In stably transfected Y-1 cells, in which the herpes simplex virus thymidine kinase (HSV-TK) gene was driven by the CYP11B1 promoter with the P450scc enhancer element, HSV-TK expression and ganciclovir sensitivity were augmented by treatment with 8-bromo-cAMP, forskolin, and ACTH. In summary, we report the construction of a suicide HSV-TK vector with preferential toxicity to adrenocortical cells. We propose that a similar strategy using differentiating agents may be useful in the gene therapy of tumors with unique differentiated properties, including those arising from other endocrine organs.
Adrenal Cortex↗
Amplification of 4q21-q22 and the MXR gene in independently derived mitoxantrone-resistant cell lines.
Molecular cytogenetic studies were conducted on three multidrug-resistant cancer sublines which are highly resistant to the chemotherapeutic agent mitoxantrone, an anthracenedione. The three independently selected sublines were derived by exposure to mitoxantrone or Adriamycin and do not overexpress MDR1 or MRP. Two sublines, MCF-7 AdVp3000 and MCF-7 MX, showed an amplification peak at 4q21-q22, as demonstrated by comparative genomic hybridization (CGH), while the third, S1-M1-80, did not. FISH using a whole chromosome 4 paint demonstrated multiple rearrangements involving chromosome 4 in MCF-7 AdVp3000 and MCF-7 MX, while S1-M1-80 contained only a simple reciprocal translocation. The parental cell lines had no chromosome 4 rearrangements and no copy number gain or amplification of chromosome 4. Spectral karyotyping (SKY) analysis revealed a balanced translocation, t(4;17)(q21-q22;p13) in S1-M1-80 and multiple clonal translocations involving chromosome 4 in MCF-7 AdVp3000 and MCF-7 MX. A novel cDNA, designated MXR, which encodes an ABC half-transporter and is highly overexpressed in the three sublines, was localized to chromosome 4 by somatic cell hybrid analysis. Southern blot analysis demonstrated amplification of the MXR gene in MCF-7 AdVp3000 and MCF-7 MX, but not in S1-M1-80. FISH studies with a BAC probe for MXR localized the gene to 4q21-22 in the normal chromosome 4 and revealed in both MCF-7 AdVp3000 and MCF-7 MX amplification of MXR at one translocation juncture, shown by SKY to be t(4;5)(4qter-->4cen-->4q21-22::5q13-->5qter++ +) in MCF-7 AdVp3000 and t(6;4;6;3)(6pter-->6q15::4q21-q22::hsr::6q?::3q?27-->+ ++3qter) in MCF MX; neither of the breakpoints in the partner chromosomes showed amplification by CGH. The data are consistent with the hypothesis of a transporter, presumably that encoded by the MXR gene, mediating mitoxantrone resistance. The MXR gene encodes a half-transporter and the absence of cytogenetic evidence of coamplification of other regions suggests that a partner may not be overexpressed, and instead the MXR half-transporter homodimerizes to mediate drug transport. Genes Chromosomes Cancer 27:110-116, 2000. Published 2000 Wiley-Liss, Inc.
Antineoplastic Agents↗
Discodermolide: just another microtubule-stabilizing agent? No! A lesson in synergy.
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Alkanes↗