Jeffrey A. Moscow

The development of multidrug resistance in MCF-7 human breast cancer cells and the acquisition of broad resistance to xenobiotics in rat hyperplastic nodules are both associated with increased P-glycoprotein (mdr) gene expression as well as changes in activities of intracellular detoxication enzymes; among these changes is a significant increase in the activity of the anionic isozyme of glutathione-S-transferase (GST). We have isolated a cDNA encoding the human anionic glutathione-S-transferase, GST pi-1, from a cDNA library constructed from multidrug-resistant MCF-7 cells. The deduced amino acid sequence of GST pi-1 shows that while the human anionic GST displays 85% nucleotide and amino acid sequence homology to the rat anionic isozyme, it is markedly less related to human basic GST isozymes. We have examined the expression of GST pi and P-glycoprotein in 170 specimens of human tissues and tumors. P-Glycoprotein RNA expression was positive in eight of 23 lymphomas and two of 12 colon tumors; however, many other normal and malignant tissues, including lung, bladder, and breast tumors, had low or undetectable levels of P-glycoprotein RNA expression. In contrast, GST pi was readily detected in a wide variety of normal and malignant tissues. The level of GST pi mRNA expression in normal tissues was heterogeneous, with lowest levels found in liver and the highest levels found in lung, esophagus, and placenta. GST pi was also variably expressed in human tumors, with the lowest relative levels occurring in lymphoma and breast cancer and the highest levels found in lung cancer and head and neck tumors. In addition, comparison of paired specimens from the same patient indicated that GST pi expression was increased in many tumors relative to matched normal tissue.

Our laboratory has previously reported the isolation of a murine cDNA which restores reduced folate carrier (RFC) activity and methotrexate (MTX) sensitivity to a MTX-resistant, transport-deficient human breast cancer cell line (MTXR ZR-75-1) (K. H. Dixon et al., J. Biol. Chem., 269: 17-20, 1994). Using this murine cDNA as a probe, we have isolated two homologous overlapping partial cDNAs from a human testis cDNA library. In addition, using human cDNA as a probe, we have isolated a 20-kb human genomic fragment which contains RFC coding regions. Analysis of the nucleotide sequence of these clones revealed that the human RFC gene, RFC1, is approximately 65% homologous to the murine and hamster genes. Using a human genomic P1 plasmid clone containing RFC1, we mapped the location of RFC1 by fluorescence in situ hybridization to the end of the long arm of chromosome 21 (21q22.2-q22.3). Fluorescence in situ hybridization analysis also showed that two copies of RFC1 were present in MTXR ZR-75-1 cells, and showed no evidence of rearrangement of this gene. Northern blot analysis of MTXR ZR-75-1 cells demonstrated a marked decrease in the level of the 3-kb RFC1 transcript relative to the parental cell line, and Western blot analysis using a polyclonal antibody raised against a peptide generated from the RFC1 sequence showed decreased expression of an approximately M(r) 56,000 protein in MTXR ZR-75-1 cells. Finally, MTXR ZR-75-1 cells transfected with an RFC1 gene showed increased MTX uptake, which was more sensitive to competition by folinic acid than by folic acid. Therefore, decreased RFC1 expression appears to be the molecular mechanism of decreased MTX uptake in this MTX-resistant cell line.

Patient-derived xenografts (PDXs) are resected human tumors engrafted into mice for preclinical studies and therapeutic testing. It has been proposed that the mouse host affects tumor evolution during PDX engraftment and propagation, affecting the accuracy of PDX modeling of human cancer. Here, we exhaustively analyze copy number alterations (CNAs) in 1,451 PDX and matched patient tumor (PT) samples from 509 PDX models. CNA inferences based on DNA sequencing and microarray data displayed substantially higher resolution and dynamic range than gene expression-based inferences, and they also showed strong CNA conservation from PTs through late-passage PDXs. CNA recurrence analysis of 130 colorectal and breast PT/PDX-early/PDX-late trios confirmed high-resolution CNA retention. We observed no significant enrichment of cancer-related genes in PDX-specific CNAs across models. Moreover, CNA differences between patient and PDX tumors were comparable to variations in multiregion samples within patients. Our study demonstrates the lack of systematic copy number evolution driven by the PDX mouse host.

The gene that produces the precursor RNA transcript to the three largest structural rRNA molecules (rDNA) is present in multiple copies and organized into gene clusters. The 10 human rDNA clusters represent <0.5% of the diploid human genome but are critically important for cellular viability. Individual genes within rDNA clusters possess very high levels of sequence identity with respect to each other and are present in high local concentration, making them ideal substrates for genomic rearrangement driven by dysregulated homologous recombination. We recently developed a sensitive physical assay capable of detecting recombination-mediated genomic restructuring in the rDNA by monitoring changes in lengths of the individual clusters. To prove that this dysregulated recombination is a potential driving force of genomic instability in human cancer, we assayed the rDNA for structural rearrangements in prospectively recruited adult patients with either lung or colorectal cancer, and pediatric patients with leukemia. We find that over half of the adult solid tumors show detectable rDNA rearrangements relative to either surrounding nontumor tissue or normal peripheral blood. In contrast, we find a greatly reduced frequency of rDNA alterations in pediatric leukemia. This finding makes rDNA restructuring one of the most common chromosomal alterations in adult solid tumors, illustrates the dynamic plasticity of the human genome, and may prove to have either prognostic or predictive value in disease progression.