Pancreatic adenocarcinoma—Genetic portrait from chromosomes to microarraysKarhu, Ritva; Mahlamäki, Eija; Kallioniemi, Anne
doi: 10.1002/gcc.20337pmid: 16688744
Pancreatic adenocarcinoma is the fifth leading cause of cancer death with a 5‐year survival rate of less than 5%. Although the role of a few known oncogenes and tumor suppressor genes in the development of pancreatic cancer is fairly well established, it is obvious that the majority of genetic changes responsible for the initiation and progression of this disease are still unknown. In this review, the authors will discuss the results from various genome‐wide screening efforts, from traditional chromosome analyses to modern DNA microarray studies, which have provided an enormous amount of information on genetic alterations in pancreatic adenocarcinoma. Exciting findings have emerged from these studies, highlighting multiple potential chromosomal regions that may harbor novel cancer genes involved in the molecular pathogenesis of this lethal disorder. These findings complete the picture of pancreatic adenocarcinoma as a genetically highly complex and heterogenous tumor type with an ongoing instability process. In addition, the precisely localized copy number changes offer a valuable starting point for further studies required to identify the genes involved and to characterize their potential functional role in the development and progression of pancreatic adenocarcinoma. © 2006 Wiley‐Liss, Inc.
Bgl II‐based panhandle and reverse panhandle PCR approaches increase capability for cloning der(II) and der(other) genomic breakpoint junctions of MLL translocationsRobinson, Blaine W.; Slater, Diana J.; Felix, Carolyn A.
doi: 10.1002/gcc.20336pmid: 16703585
Panhandle PCR techniques to amplify known sequence flanked by unknown sequence have been useful for MLL genomic breakpoint junctions and fusion transcripts because MLL has a large number of partner genes. However, genomic panhandle PCR approaches are impeded when the restriction fragment that contains the breakpoint junction is too large to amplify. We devised new panhandle PCR approaches for MLL genomic breakpoint junctions that create the template from BglII restriction fragments by attaching MLL sequence to a BglII site in the partner gene. This leads to the annealing of MLL and its complement in the handle and creates an intrastrand loop containing the breakpoint junction sequence for amplification with primers all from MLL. BglII panhandle PCR for der(11) breakpoint junctions was accomplished by ligating a phosphorylated oligonucleotide containing a BglII overhang and sequence complementary to MLL exon 7 to the 3′ ends of BglII digested DNA, and forming the template from the sense strand of DNA. In BglII reverse panhandle PCR for der(other) breakpoint junctions, a phosphorylated oligonucleotide containing a BglII overhang and the complement of antisense sequence in MLL exon 10 was ligated to the 3′ ends of BglII digested DNA, and the template was formed from the antisense strand of DNA. These approaches amplified 5′‐MLL‐MLLT4‐3′ and 5′‐AFF1‐MLL‐3′ breakpoint junctions. The former is significant because few t(6;11) genomic breakpoint junctions have been sequenced. BglII panhandle PCR approaches increase the possibilities for cloning MLL genomic breakpoint junctions where there is heterogeneity in partner genes and breakpoint locations. © 2006 Wiley‐Liss, Inc.
MLL chimeric protein activation renders cells vulnerable to chromosomal damage: An explanation for the very short latency of infant leukemiaEguchi, Mariko; Eguchi‐Ishimae, Minenori; Knight, Deborah; Kearney, Lyndal; Slany, Robert; Greaves, Mel
doi: 10.1002/gcc.20338pmid: 16688745
MLL fusion genes are a predominant feature of acute leukemias in infants and in secondary acute myeloid leukemia (AML) associated with prior chemotherapy with topo‐II poisons. The former is considered to possibly arise in utero via transplacental chemical exposure. A striking feature of these leukemias is their malignancy and remarkably brief latencies implying the rapid acquisition of any necessary additional mutations. We have suggested that these coupled features might be explained if MLL fusion gene encoded proteins rendered cells more vulnerable to further DNA damage and mutation in the presence of chronic exposure to the agent(s) that induced the MLL fusion itself. We have tested this idea by exploiting a hormone regulated MLL‐ENL (MLLT1) activation system and show that MLL‐ENL function in normal murine progenitor cells substantially increases the incidence of chromosomal abnormalities in proliferating cells that survive exposure to etoposide VP‐16. This phenotype is associated with an altered pattern of cell cycle arrest and/or apoptosis. © 2006 Wiley‐Liss, Inc.
RNA interference‐based functional dissection of the 17q12 amplicon in breast cancer reveals contribution of coamplified genesKao, Jessica; Pollack, Jonathan R.
doi: 10.1002/gcc.20339pmid: 16708353
DNA amplification is a frequent occurrence in cancer genomes. While tumor amplicons may harbor known oncogenes “driving” amplification, amplicons rarely comprise only single genes. The potential functional contribution of coamplified genes remains largely unexplored. In breast cancer, 20–30% of tumors exhibit amplification within chromosome band 17q12, containing the ERBB2 oncogene. Analysis of array‐based comparative genomic hybridization and expression profiling data indicate that the minimum region of recurrent amplification (i.e., the amplicon “core”) at 17q12 includes two other genes, GRB7 and STARD3, which exhibit elevated expression when amplified. Western blot analysis confirms overexpression of each at the protein level in breast cancer cell lines SKBR3 and BT474 harboring amplification. In these cell lines (but not in control MCF7 breast cancer cells lacking 17q12 amplification), targeted knockdown of ERBB2 expression using RNA interference (RNAi) methods results in decreased cell proliferation, decreased cell‐cycle progression, and increased apoptosis. Notably, targeted knockdown of either GRB7 or STARD3 also leads to decreased cell proliferation and cell‐cycle progression, albeit to a lesser extent compared with ERBB2 knockdown. We conclude that the amplification and resultant overexpression of genes coamplified with ERBB2 at 17q12 can contribute to proliferation levels of breast cancer cells. Our findings validate the utility of RNAi in the functional interrogation of tumor amplicons, and provide evidence for a contribution of coamplified genes to tumor phenotypes. © 2006 Wiley‐Liss, Inc.
Mutational dynamics in human tumors confirm the neutral intrinsic instability of the mitochondrial D‐loop poly‐cytidine repeatSchwartz, Simó; Alazzouzi, Hafid; Perucho, Manuel
doi: 10.1002/gcc.20340pmid: 16708351
Somatic mutations at a mitochondrial noncoding polycytidine (C)n repeat (polyC) have been associated with tumor progression. We analyzed whether these alterations are due to the inherent mutability of repeated sequences. Insertion and deletion mutations were found in colon (n = 114), stomach (n = 105), endometrium (n = 53), breast (n = 45), lung (n = 35), and prostate (n = 20) tumors. The mutation frequency in colon, gastric, and endometrial tumors was 23, 17, and 11%, respectively, which paralleled the relative extent of microsatellite instability in long mononucleotide repeats observed in tumors with mismatch repair deficiency (colon > stomach > endometrium, relative ratio 10:8:4). Colon tumors with mutations of more than one nucleotide were more advanced in tumor progression. Further, two tumors showing a T > C mutation that restored the homopolymeric repeat, harbored sequential deletion mutations of up to 4 and 6 nucleotides. These results illustrate that the increased mutability of repeated mitochondrial sequences is dependent on the repetitive structure of the DNA molecule and suggest that mutations in the (C)n repeat, whether homoplasmic or not, and by extrapolation, mitochondrial mutations in general, are not the result of selective pressure during tumorigenesis. We also suggest that the (C)n repeat may be used as an universal molecular clock to estimate the relative mitotic history of tumors. © 2006 Wiley‐Liss, Inc.