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HTI-286, a synthetic analogue of hemiasterlin, depolymerizes microtubules and is proposed to bind at the Vinca peptide site in tubulin. It has excellent in vivo antitumor activity in human xenograft models, including tumors that express P-glycoprotein, and is in phase II clinical evaluation. To identify potential mechanisms of resistance induced by HTI-286, KB-3-1 epidermoid carcinoma cells were exposed to increasing drug concentrations. When maintained in 4.0 nmol/L HTI-286, cells had 12-fold resistance to HTI-286. Cross-resistance was observed to other Vinca peptide-binding agents, including hemiasterlin A, dolastatin-10, and vinblastine (7- to 28-fold), and DNA-damaging drugs, including Adriamycin and mitoxantrone (16- to 57-fold), but minimal resistance was seen to taxanes, epothilones, or colchicine (1- to 4-fold). Resistance to HTI-286 was retained when KB-HTI-resistant cells were grown in athymic mice. Accumulation of [3H]HTI-286 was lower in cells selected in intermediate (2.5 nmol/L) and high (4.0 nmol/L) concentrations of HTI-286 compared with parental cells, whereas accumulation of [14C]paclitaxel was unchanged. Sodium azide treatment partially reversed low HTI-286 accumulation, suggesting involvement of an ATP-dependent drug pump. KB-HTI-resistant cells did not overexpress P-glycoprotein, breast cancer resistance protein (BCRP/ABCG2/MXR), MRP1, or MRP3. No mutations were found in the major β-tubulin isoform. However, 4.0 nmol/L HTI-286-selected cells had a point mutation in α-tubulin that substitutes Ser for Ala12 near the nonexchangeable GTP-binding site of α-tubulin. KB-HTI-resistant cells removed from drug became less resistant to HTI-286, no longer had low HTI-286 accumulation, and retained the Ala12 mutation. These data suggest that HTI-286 resistance may be partially mediated by mutation of α-tubulin and by an ATP-binding cassette drug pump distinct from P-glycoprotein, ABCG2, MRP1, or MRP3.

Reports of multiple distinct mitoxantrone-resistant sublines without overexpression of P-glycoprotein or the multidrug-resistance associated protein have raised the possibility of the existence of another major transporter conferring drug resistance. In the present study, a cDNA library from mitoxantrone-resistant S1-M1-80 human colon carcinoma cells was screened by differential hybridization. Two cDNAs of different lengths were isolated and designated MXR1 and MXR2. Sequencing revealed a high degree of homology for the cDNAs with Expressed Sequence Tag sequences previously identified as belonging to an ATP binding cassette transporter. Homology to the Drosophila white gene and its homologues was found for the predicted amino acid sequence. Using either cDNA as a probe in a Northern analysis demonstrated high levels of expression in the S1-M1-80 cells and in the human breast cancer subline, MCF-7 AdVp3000. Levels were lower in earlier steps of selection, and in partial revertants. The gene is amplified 10-12-fold in the MCF-7 AdVp3000 cells, but not in the S1-M1-80 cells These studies are consistent with the identification of a new ATP binding cassette transporter, which is overexpressed in mitoxantrone-resistant cells.

A cDNA encoding the novel drug resistance gene, LRP (originally termed lung resistance-related protein), was isolated from HT1080/DR4, a 220-fold doxorubicin-resistant human fibrosarcoma cell line which displays a multidrug resistance phenotype and overexpresses the multidrug resistance protein (MRP) but does not overexpress P-glycoprotein encoded by the MDR1 gene, Using the full-length 2.8-kb cDNA probe, the gene for LRP was regionally localized to the 16p13.1-16p11.2 chromosomal segment in human metaphases. Dual color fluorescence in situ hybridization studies refined the localization of LRP to 16p11.2, a location approximately 27 cM proximal to MRP (16p13.1). Two color hybridization studies indicated that HT1080/DR4 fibrosarcoma cells contain amplification of both the MRP and LRP genes in a striking striped pattern in the homogeneously staining region, hsr(7)(p12p15). in contrast, only amplified MRP gene sequences were contained within the homogeneously staining region, hsr(18q). Amplification of LRP was not identified in any of seven other drug-resistant tumor cell lines characterized by 20-300-fold levels of doxorubicin resistance, including two cell lines known to overexpress LRP (SW1573/2R120 and GLC4/ADR). Amplified MRP gene sequences mere identified in H69AR, GLC4/ADR, and HL-60/AR whereas only MDR1 gene amplification was observed in the S1B120 colon carcinoma cell line. These data indicate that although both the MRP and LRP genes map to the short arm of chromosome 16, they are rarely coamplified and are not normally located within the same amplicon. A key role for chromosome breakage in gene amplification is supported by the presence of non-random karyotypic anomalies near the MRP and LRP normal cellular loci.