Lyn A. Mickley

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.

Cytogenetic and molecular characterization of random chromosomal rearrangements activating the drug resistance gene, MDR1/P-glycoprotein, in drug-selected cell lines and patients with drug refractory ALL

Drug resistance, both primary and acquired, is a major obstacle to advances in cancer chemotherapy. In vitro, multidrug resistance can be mediated by P‐glycoprotein (PGY1), a cell surface phosphoglycoprotein that acts to efflux natural products from cells. PGY1 is encoded by the MDR1 gene located at 7q21.1. Overexpression of MDR1 has been demonstrated in many cancers, both in patient tumors and in cell lines selected with a variety of chemotherapeutic agents. Recent studies in drug‐selected cell lines and patients samples have identified hybrid mRNAs comprised of an active, but apparently random, gene fused 5′ to MDR1. This observation indicates that random chromosomal rearrangements, such as translocations and inversions, leading to “capture” of MDR1 by constitutively expressed genes may be a mechanism for activation of this gene following drug exposure. In this study, fluorescence in situ hybridization (FISH) using whole chromosome paints (WCP) and bacterial artificial chromosome (BAC)‐derived probes showed structural rearrangements involving 7q in metaphase and interphase cells, and comparative genomic hybridization (CGH) revealed high levels of amplification at chromosomal breakpoints. In an adriamycin‐selected resistant colon cancer line (S48–3s/Adr), WCP4/WCP7 revealed t(4;7)(q31;q21) and BAC‐derived probes demonstrated that the breakpoint lay between MDR1 and sequences 500–1000 KB telomeric to it. Similarly, in a subline isolated following exposure to actinomycin D (S48–3s/ActD), a hybrid MDR1 gene composed of heme oxygenase‐2 sequences (at 16p13) fused to MDR1 was identified and a rearrangement confirmed with WCP7 and a subtelomeric 16p probe. Likewise, in a paclitaxel‐selected MCF‐7 subline where CASP sequences (at 7q22) were shown to be fused to MDR1, WCP7 showed an elongated chromosome 7 with a homogeneously staining regions (hsr); BAC‐derived probes demonstrated that the hsr was composed of highly amplified MDR1 and CASP sequences. In all three selected cell lines, CGH demonstrated amplification at breakpoints involving MDR1(at 7q21) and genes fused to MDR1 at 4q31, 7q22, and 16p13.3. Finally, in samples obtained from two patients with drug refractory ALL, BAC‐derived probes applied to archived marrow cells demonstrated that a breakpoint occurred between MDR1 and sequences 500–1000 KB telomeric to MDR1, consistent with a random chromosomal rearrangement. These results support the proposal that random chromosomal rearrangement leading to capture and activation of MDR1 is a mechanism of acquired drug resistance. Genes Chromosomes Cancer 23:44–54, 1998.

An ATP-binding cassette gene (ABCG3) closely related to the multidrug transporter ABCG2 (MXR/ABCP) has an unusual ATP-binding domain

The recent identification of multiple ABC transporters with potential roles in drug resistance has offered hope for the treatment of cancer (Borst 1999; Sandor et al. 1998). ATP binding cassette (ABC) proteins bind and hydrolyze ATP providing energy for the transport of an array of substrates (Dean and Allikmets 1995; Higgins 1992). MDR1/P-glycoprotein (ABCB1) was the first transporter described in association with drug resistance, and inhibitors of P-glycoprotein-mediated transport are undergoing clinical trials for reversal of drug resistance. However, numerous laboratory models have been described with non-ABCB1-mediated drug resistance. Eukaryotic ABC transporters are either full size, as are ABCB1, and the MRPs, with 12 transmembrane domains and two ATP binding sites on each molecule; or they are half-size with six transmembrane domains and one ATP binding site (Dean and Allikmets 1995). The half-transporters dimerize to form a functional transporter (Ewart and Howells 1998; Shani and Valle 1998). Dimerization could allow for diversification in the function of ABC transporters by increasing the number of possible combinations. Recently a half-transporter molecule, MXR/ABCP/ ABCG2, was identified in cell lines with non-ABCB1-mediated multidrug resistance characterized by high levels of resistance to mitoxantrone, anthracyclines, and to the camptothecin analogs, topotecan, SN38, and 9AC (Allikmets et al. 1998; Brangi et al. 1999; Doyle et al. 1998; Maliepaard et al. 1999; Miyake et al. 1999; Litman et al. 2000). This protein is a half-transporter with six transmembrane domains and one ATP binding site, and bears striking homology to theDrosophilawhite genes. Computer searches of the EST databases with the BLAST program led to the identification of several mouse and rat sequences that had high homology to ABCG2 but that appeared to encode a unique gene. RACE was used to amplify and clone the entire coding region of the gene by using mouse spleen RNA, and the sequence revealed a single open reading frame encoding a 650-AA protein, designated Abcg3. Primers to the 3 8 untranslated region were used to isolate a BAC clone from a mouse 129SV library. Each exon of the gene was sequenced from the BAC clone to identify the splice junction sites. The Abcg3gene has 16 exons, including one 58 non-coding exon. Figure 1 displays an alignment of Abcg3 with the amino acid sequence of the other white family genes. Considerable identity is seen in the ATP-binding domain, but clear homology is seen throughout the coding region. Abcg3 is most closely related to ABCG2 with 54% amino acid identity overall, 64% in the NBF and 50% in the TM region. The alignment was used to generate a phylogenetic tree of the genes, and this analysis confirms that Abcg3 and ABCG2 are closely related (data not shown). Surprisingly, Abcg3 contains several unusual residues in the Walker A and signature (C) domains. The Walker A consensus is GAGKST, and the Abcg3 sequence is DGSRSL; the C region consensus is LSGG, and the Abcg3 sequence in this region is RSKE. Many of these residues are absolutely conserved in all ABC genes, and mutations in these regions typically lead to non-functional proteins. This suggests that Abcg3 may not bind and/or hydrolyze ATP. The Abcg3BAC clone was used for in situ hybridization on both mouse and human chromosomes. The gene localized to a single region on mouse Chr 5, band E3-4. The murine BAC clone gave a single signal on human Chr 8p12 (data not shown). However, degenerate PCR and database searches have failed to reveal a human Abcg2-related gene. The mouse Abcg3 gene was also mapped by radiation hybrid analysis to mouse Chr 5, 59 cM from the centromere, a position consistent with the in situ hybridization data (Schriml and Dean 2000). Using a quantitative PCR assay previously developed for evaluating MDR1 (multidrug resistance-1) expression, we examined expression of Abcg2andAbcg3in normal murine tissues. The levels of expression in the various tissues were quantitated as previously described for MDR1 and normalized to the level found in muscle, which was arbitrarily assigned a value of 10. Muscle expression ofAbcg2andAbcg3was at low but detectable levels. Figure 2A presents a bar graph depicting the PCR expression data. The highest levels of expression for Abcg2 were found in the kidney, lung, and small intestine. For Abcg3,the highest levels of expression were found in thymus and spleen. Except for the relative higher levels of expression in the small intestine for both Abcg2andAbcg3,there was no concordance in expression pattern. To confirm the pattern of Abcg3expression, Northern analysis was performed and is shown in Fig. 2B. Although the tissues with the lower levels of expression are below the level of detection by Northern blot analysis, the high levels found in the thymus and spleen are still detectable; with faint signals also detected in lung and small intestine. It should be noted that the quantitative values were calculated from PCR product generated in the exponential range of amplification. The ABCG2 gene is amplified and/or overexpressed in several multidrug resistant tumor cell lines derived from breast, colon, stomach, and leukemic cells (Doyle et al. 1998; Miyake et al., 1999). The cells overexpressing ABCG2 are resistant to mitoxantrone, bisantrene, anthracyclines, and the camptothecin analogs * Present address: National Center for Biotechnology Information, NLM, NIH, Bethesda, MD 20892, USA

Modulation of EGF receptor expression by differentiating agents in human colon carcinoma cell lines.

The existence of an autocrine loop for self-stimulation of growth in malignant cells has been proposed for transforming growth factor-alpha (TGF alpha) and its receptor, the epidermal growth factor (EGF) receptor, in a variety of malignant cell types. Expression of both has been described in colon carcinoma. In order to investigate whether there is a correlation between TGF alpha and EGF receptor mRNA expression and differentiation, we studied the effects of differentiating agents on seven human colon carcinoma cell lines. All of the lines responded to the differentiating agents. In four of the seven lines there was increased EGF receptor mRNA two to five days after treatment with 2 mM sodium butyrate. In three of these lines TGF alpha mRNA and protein were also increased. In the one cell line treated with the differentiating agents DMF and DMSO, EGF receptor mRNA was also increased. [125I]-EGF binding to the cells was measured before and after treatment with butyrate. In two of three cell lines, increased EGF receptor mRNA was accompanied by a 2.4-fold increase in the number of binding sites per cell. In SW620 cells, no EGF receptor binding was detected before or after butyrate treatment. In the two cell lines in which butyrate increased EGF receptor binding, simultaneous treatment with EGF did not enhance growth. These data demonstrate increased expression of the TGF alpha/EGF receptor system after differentiation of colon carcinoma cell lines and suggest that their expression may be characteristic of a differentiated phenotype.

INTRINSIC DRUG RESISTANCE IN PRIMARY AND METASTATIC RENAL CELL CARCINOMA

Much remains to be learned about drug resistance in the biology of RCC and its metastases. We measured MDR-1/P-glycoprotein expression in 19 tumor samples from patients with metastatic RCC by RNase protection and quantitative PCR assays. The median level of the 16 tumor metastases was 4.9 (range: 0.10 to 156.2) relative to the level of 10 assigned to a reference cell line, SW620, which has been characterized as expressing a minimum level of MDR-1. Since these levels were lower than expected for RCC, we asked whether the metastases possessed a phenotype different from primary RCC and examined MDR-1 expression in 5 paired cell lines derived from primary and metastatic RCC. In 8/10 lines, MDR-1 expression was >10. Relative to the level in the primary line, MDR-1 expression was decreased (3 to 50-fold) in 3 metastatic lines, was increased in 1, and unchanged in 1. MRP mRNA expression was lower in the metastatic lines while EGFR expression was variable. IC50 values for 6 compounds (including 4 standard agents and one new Phase 1 agent) were determined for the paired lines. Rhodamine and calcein efflux assays were performed as measures of P-glycoprotein and MRP function. Rhodamine efflux correlated with MDR-1 mRNA expression (r = 0.87) and with the IC50s (r = 0.60) for paclitaxel in the paired cell lines. In contrast, calcein efflux did not correlate with MRP expression. Lastly, MDR-1 expression correlated with cytokeratin 8 (CK8) protein levels, a measure of cellular differentiation. In sum, these data suggest renal cell carcinoma (RCC) metastases have altered MDR-1 expression potentially due to altered differentiation relative to the primary tumor. Thus, the drug resistance phenotype of primary RCC tumors may not reflect that of their metastases.

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 λ 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 χ‐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. Int. J. Cancer 86:506–511, 2000.

Cytogenetic, spectral karyotyping, fluorescence in situ hybridization, and comparative genomic hybridization characterization of two new secondary leukemia cell lines with 5q deletions, and MYC and MLL amplification.

Cytogenetic studies of patients with therapy‐induced acute myeloid leukemia (t‐AML) have demonstrated whole chromosome loss or q‐arm deletion of chromosomes 5 and/or 7 in a majority of cases. We have established two cell lines, SAML‐1 and SAML‐2, from two patients who developed t‐AML after radiation and chemotherapy for Hodgkin disease. In both cases, the leukemia cells contained 5q deletions. SAML‐1 has 58 chromosomes and numerous abnormalities, including der(1)(1qter→1p22::5q31→5qter), der(5)(5pter→5q22::1p22→1pter), +8, der(13)i(13)(q10)del(13)(q11q14.1), and t(10;11). Fluorescence in situ hybridization (FISH) with unique sequence probes for the 5q31 region showed loss of IL4, IL5, IRF1, and IL3, and translocation of IL9, DS5S89, EGR1, and CSFIR to 1p. SAML‐2 has 45 chromosomes, del(5)(q11.2q31) with a t(12;13)ins(12;5), leading to the proximity of IRF1 and RB1, and complex translocations of chromosomes 8 and 11, resulting in amplification of MYC and MLL. Comparative genomic hybridization and spectral karyotyping were consistent with the G‐banding karyotype and FISH analyses. Because a potential tumor suppressor(s) in the 5q31 region has yet to be identified, these cell lines should prove useful in the study of the mechanisms leading to the development of t‐AML.