Ilana Cohen

Clinical implications of UGT1A1*28 genotype testing in colorectal cancer patients

Metastatic colorectal cancer is frequently treated with irinotecan, a topoisomerase‐I inhibitor. The UGT1A1 gene encodes for an enzyme that metabolizes irinotecan, and its genetic variants were shown to be associated with increased drug toxicity. We evaluated clinical outcomes associated with the UGT1A1*28 variant.

TBP flanking sequences: asymmetry of binding, long-range effects and consensus sequences.

We carried out in vitro selection experiments to systematically probe the effects of TATA-box flanking sequences on its interaction with the TATA-box binding protein (TBP). This study validates our previous hypothesis that the effect of the flanking sequences on TBP/TATA-box interactions is much more significant when the TATA box has a context-dependent DNA structure. Several interesting observations, with implications for protein–DNA interactions in general, came out of this study. (i) Selected sequences are selection-method specific and TATA-box dependent. (ii) The variability in binding stability as a function of the flanking sequences for (T-A)4 boxes is as large as the variability in binding stability as a function of the core TATA box itself. Thus, for (T-A)4 boxes the flanking sequences completely dominate and determine the binding interaction. (iii) Binding stabilities of all but one of the individual selected sequences of the (T-A)4form is significantly higher than that of their mononucleotide-based consensus sequence. (iv) Even though the (T-A)4 sequence is symmetric the flanking sequence pattern is asymmetric. We propose that the plasticity of (T-A)n sequences increases the number of conformationally distinct TATA boxes without the need to extent the TBP contact region beyond the eight-base-pair long TATA box.

A dominant-negative form of POM121 binds chromatin and disrupts the two separate modes of nuclear pore assembly

Nuclear pore complexes (NPCs) are formed during two separate stages of the metazoan cell cycle. They are assembled into the re-forming nuclear envelope (NE) at the exit from mitosis and into an intact, expanding NE during interphase. Here, we show that a soluble internal fragment of the membrane nucleoporin POM121 has a dominant-negative effect on both modes of assembly in a cell-free reconstitution system. The soluble POM121 fragment binds chromatin at sites that are distinct from ELYS–Nup107–160 ‘seeding’ sites and prevents membrane enclosure and NPC formation. Importin-β negatively regulates chromatin binding by the POM121 fragment through a conserved NLS motif and is also shown to affect the recruitment of the endogenous membrane protein to chromatin in the full assembly system. When an intact NE is present before the addition of the dominant-negative fragment, NPCs are inserted into the NE but membrane expansion is inhibited. This results in densely packed NPCs with no intervening membrane patches, as visualized by scanning electron microscopy. We conclude that POM121 plays an important role in both modes of assembly and links nuclear membrane formation and expansion to nuclear pore biogenesis.

MutYH mutation carriers have increased breast cancer risk

Variants of the mutY homolog gene MutYH, a DNA repair gene, are associated with increased risk of colorectal cancer; however, it remains unclear whether these variants also are associated with the risk of other cancers. The authors studied the risk of breast cancer associated with MutYH variants in a unique ethnic group of Sephardi Jews in Israel with a high prevalence of MutYH mutations.

Nuclear import of an intact preassembled proteasome particle

Nuclear targeting of intact proteasome particles was tested in the Xenopus egg extract system. Both the 26S proteasome holoenzyme and the 20S core particle were targeted to the nuclear envelope but could not enter the nucleus. A novel proteolytically active 20S+ particle was actively imported into the nucleoplasm in a Ran-independent fashion.

Targeting of Viral Capsids to Nuclear Pores in a Cell-Free Reconstitution System

Many viruses deliver their genomes into the nucleoplasm for viral transcription and replication. Here, we describe a novel cell‐free system to elucidate specific interactions between viruses and nuclear pore complexes (NPCs). Nuclei reconstituted in vitro from egg extracts of Xenopus laevis, an established biochemical system to decipher nuclear functions, were incubated with GFP‐tagged capsids of herpes simplex virus, an alphaherpesvirus replicating in the nucleus. Capsid binding to NPCs was analyzed using fluorescence and field emission scanning electron microscopy. Tegument‐free capsids or viral capsids exposing inner tegument proteins on their surface bound to nuclei, while capsids inactivated by a high‐salt treatment or covered by inner and outer tegument showed less binding. There was little binding of the four different capsid types to nuclei lacking functional NPCs. This novel approach provides a powerful system to elucidate the molecular mechanisms that enable viral structures to engage with NPCs. Furthermore, this assay could be expanded to identify molecular cues triggering viral genome uncoating and nuclear import of viral genomes.

Abstract 2701: Clinical implications of UGT1A1*28 genotype testing in colorectal cancer patients

Purpose: Metastatic colorectal cancer is frequently treated with Irinotecan (CPT-11), a topoisomerase-I inhibitor. Irinotecan toxicity is relatively commonly observed. The gene UGT1A1 encodes an enzyme that catalyzes the glucuronidation of the active irinotecan metabolite SN-38 which is eliminated in the liver by metabolic alteration to an inactive form SN-38G. We retrospectively evaluated the association between UGT1A1 genetic variation, prevalence of severe toxicity and colorectal survival. Patients and Methods: In the Molecular Epidemiology of Colorectal Cancer (MECC) Study we identified 213 patients with metastatic colorectal cancer treated by irinotecan-based chemotherapy (FOLFIRI, IFL). DNA extracted from blood was used for genotyping either by simple determination of TATA box sequence in patient DNA or by implementation of fragment analysis that detects the difference in the number of nucleotides in the specific region analyzed. The polymorphism UGT1A1*28 is characterized by the presence of an additional TA repeat in the TATA sequence of the UGT1A1 promoter, ((TA)7TAA, instead of (TA)6TAA). Data on side effects and survival were extracted from full follow-up oncology records. Results: UGT1A1*28 genotypes 6/6, 6/7, 7/7 were detected in 41.8%, 46.5%, and 11.7% of the patients respectively. Observed grade 3-4 toxicity included: diarrhea 22.4%, leucopenia 9.3%, neutropenia 8.9%, neutropenic fever 3.7%, and mucositis 4%. No significant difference was observed in the rate of diarrhea in patients with UGT1A1*28 7/7 genotype (20%) as compared to these with the 6/6 genotype (27.5%). Hematological toxicity was significantly more common in patients with the 7/7 genotype. Neutropenic fever was observed in 24% of the 7/7 genotype but only in 2% of the 6/7 genotype and 0% of the 6/6 genotype. Oncological hospitalization rate was very high among the 7/7 group (45.8%) and significantly higher than among the 6/6 group (14.4%). The Median overall survival of patients with 7/7 was significantly lower (2.10 years) than that of patients with the 6/6 variant (4.13 years, p=0.002). Conclusion: UGT1A1*28 7/7 genotype is strongly associated with severe toxicity (mainly neutropenic fever) and oncological hospitalizations and with lower survival. These data support the FDA recommendation and product labeling to tailor treatment plans for colorectal cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2701.