Fhit Interaction with Ferredoxin Reductase Triggers Generation of Reactive Oxygen Species and Apoptosis of Cancer Cells

Abstract

Fhit protein is lost in most cancers, its restoration suppresses tumorigenicity, and virus-mediated FHIT gene therapy induces apoptosis and suppresses tumors in preclinical models. We have used protein cross-linking and proteomics methods to characterize a Fhit protein complex involved in triggering Fhit-mediated apoptosis. The complex includes Hsp60 and Hsp10 that mediate Fhit stability and may affect import into mitochondria, where it interacts with ferredoxin reductase, responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin. Viral-mediated Fhit restoration increases production of intracellular reactive oxygen species, followed by increased apoptosis of lung cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape apoptosis, carrying serious oxidative DNA damage that may contribute to an increased mutation rate. Characterization of Fhit interacting proteins has identified direct effectors of the Fhit-mediated apoptotic pathway that is lost in most cancers through loss of Fhit.

Intramitochondrial calcium regulation by the FHIT gene product sensitizes to apoptosis

“…Our results bring this tumor suppressor to the core mechanism of the intrinsic pathway of apoptosis, participating in the regulation of the critical mitochondrial steps. This effect is mediated by the fraction of Fhit localized in mitochondria (8), whereas other reported functions of Fhit, such as the putatively transcriptional control of the cell cycle are unaffected by an exclusively mitochondrial Fhit chimera. These data thus suggest that the complex intracellular distribution of Fhit may underlie a synergistic effect of different protein pools (cell cycle block, induction of apoptosis) that cooperate in repairing or eventually clearing DNA-damaged cells.…”

“…Fhit is partly localized in mitochondria, and interaction with Hsp60/ Hsp10 could be important for correct refolding after import and Fhit stability (8). This compartmentalization of Fhit could reveal a transcription-independent regulation of cell fate.…”

Virus-Free Synthesis of a Hepatitis C Virus P7 cDNA through a Three- Steps Polymerase Chain Reaction

“…More specifically, here we describe the development of this new approach by successfully synthesizing a cDNA corresponding to the HCV p7 sequence. Experiments are in progress in our laboratory to generate expression vectors carrying both wild-type and tagged p7 sequences to be used for the isolation of p7-interacting proteins in a proteomic-based approach [20] from liver eukaryotic cell systems. This will help us to shed light on the molecular mechanisms governing p7 activity in the pathogenesis of HCV-driven hepatitis.…”

“…p7 is characterized by an elevated genetic variability, a high content of hydrophobic amino acids as well as the small size; therefore, gene amplification and protein expression is difficult to achieve by wellknown methodologies [12]. Different studies to obtain adequate p7 amount to determine functional and structural features have been carried out [13][14][15][16][17][18][19][20]. Nevertheless these studies revealed crucial steps, such as RNA instability and the presence of inhibitory substances, especially in biological samples, which represent the greatest problems to be overcome [4].…”

A knockdown with smoke model reveals FHIT as a repressor of Heme oxygenase 1

“…4,6,20 Recent experiments demonstrate that Fhit deficiency promotes epithelial-mesenchymal transition, that FHIT expression protects genome integrity after carcinogen treatment, and also establish a role for Fhit in mitochondrial biology. [21][22][23][24][25][26][27] Hanahan and Weinberg summarized that cancer cells are marked by the gain of 6 abnormal behaviors. 12,14,28 Genome instability and subsequent genetic alterations underlie the acquisition of the hallmarks of cancer, as successive alterations in DNA produce genotypes that confer selective advantages.…”