1. ¹Department of Clinical Oncology, Sir YK Pao Centre for Cancer, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China
2. ²Department of Anatomical and Cellular Pathology, Sir YK Pao Centre for Cancer, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China
3. ³Department of Surgery, Sir YK Pao Centre for Cancer, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China
4. ⁴Departments of Medical Biophysics and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5G 2L9
5. ⁵Microarray Centre, Clinical Genomics Centre, University Health Network, Toronto, Ontario, Canada M5G 2C4
6. ⁶Oncology Centre, Hong Kong Sanatorium & Hospital, Hong Kong SAR, China

Correspondence: Dr N Wong, DPhil, Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, NT, Hong Kong, China. E-mail: natwong@cuhk.edu.hk

Received 3 September 2004; Revised 11 January 2005; Accepted 11 January 2005; Published online 14 March 2005.

Abstract

Administration of doxorubicin has been shown to prolong survival of patients with hepatocellular carcinoma (HCC). However, treatment regimen is often complicated by the emergence of drug resistance. The goal of our study is to enhance our understanding on the genetic changes that confer cellular chemoresistance to doxorubicin. To model this insensitive response, we established five doxorubicin-resistant (DOR) sublines through repeated exposure of escalating doses of doxorubicin to HCC cell lines (HKCI-2, -3, -4, -C1 and -C2). The DOR sublines developed displayed an average 17-fold higher IC₅₀ value than their sensitive parental cell lines. The resistant phenotype displayed was investigated by the genome-wide analyses of comparative genomic hybridization (CGH) and complementary DNA microarray for the affected genomic anomalies and deregulated genes expressed, respectively. Over-representations of regional chr. 7q11–q21, 8q22–q23 and 10p13–pter were indicated in the DOR sublines from CGH analysis. Of particular interest was the finding of amplicon augmentations from regional or whole chromosome gains during the clonal expansion of resistant sublines. Most notably, recurring amplicon 7q11.2–q21 identified coincided with the location of the multi-drug-resistant gene, MDR1. The potential involvement of MDR1 was examined by quantitative reverse transcription-polymerase chain reaction RT-PCR (qRT-PCR), which indicated an upregulation in all DOR sublines (P=0.015). Consistent overexpression of the translated MDR1 gene, P-glycoprotein, in all five DOR sublines was further confirmed in Western blot analysis. Two distinct cluster dendrograms were achieved between the DOR sublines and their sensitive parental counterparts in expression profiling. Within the doxorubicin-resistant group, distinct features of candidate genes overexpressions including ABC transporting proteins, solute carriers and TOP2A were suggested. Further assessment of TOP2A messenger RNA levels by qRT-PCR confirmed array findings and pinpointed to a common up-regulation of TOP2A in DOR sublines. Our present study highlighted areas of genomic imbalances and candidate genes in the acquired doxorubicin-resistance behavior of HCC cells.