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Methods for identifying fragile histidine triad (Fhit) interaction and uses thereof

A triplet, histidine technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve problems such as Fhit's little knowledge

Inactive Publication Date: 2011-07-27
THE OHIO STATE UNIV RES FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although Fhit expression triggers apoptosis in several experimental models through caspase-dependent mechanisms involving extrinsic and intrinsic apoptotic pathways, the early events of this process and how Fhit loss is involved in tumor initiation are still poorly understood little

Method used

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  • Methods for identifying fragile histidine triad (Fhit) interaction and uses thereof
  • Methods for identifying fragile histidine triad (Fhit) interaction and uses thereof
  • Methods for identifying fragile histidine triad (Fhit) interaction and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment I

[0111] Materials and methods

[0112] Cells, Vectors and Antisera - A549, H1299, MKN74-E4 and A116 and HCT116 cells were maintained in RPMI 1640 medium plus 10% fetal bovine serum and penicillin / streptomycin (Sigma). HEK293 cells (Microbix) used to prepare recombinant adenoviruses were cultured in Dulbecco's modified Eagle's medium plus 10% fetal calf serum and penicillin / streptomycin. AdFHIT-His6 virus was prepared as described in Example II herein. [His6-SEQ ID NO: 32] [penta-His-SEQ ID NO: 33].

[0113] Full-length FDXR was cloned from human brain cDNA (Clontech), subcloned into the pcDNA3.1 / V5-HisTOPO TA vector (Invitrogen) and sequenced; details are described in the accompanying methods below. Using Lipofectamine TM (Invitrogen) cells were transfected according to the manufacturer's instructions.

[0114] Western blot analysis - Western blot analysis was performed as described in (13) using: monoclonal anti-pentaHis (Qiagen); rabbit polyclonal anti-Fhit (Zymed Laborat...

Embodiment III

[0124] Production of Recombinant Adenovirus - Recombinant adenovirus carrying wild-type FHIT cDNA (AdFHIT) was prepared as previously described (Ishii et al., 2001 Cancer Res 61: 1578-1584). His-tagged FHIT cDNA was generated by PCR using the following oligonucleotides: 5'-ACgTggATCCCTgTgAggACATgTCgTTCAgATTTggC-3' (forward) [SEQ ID NO: 26] and 5'-TTgTggATCCTTATCAgTgATggTgATggTgATgCgATCCTCTCTgAAAgTAgCCCgCAg-3' [SHQ ID NO: 27]. These primers were designed with a BamHI restriction site for subcloning into the transfer vector pAdenoVator-CMV5-IRES-GFP. Ad-His 6 is using AdenoVator TM Kits (Qbiogene, Carlsbad, CA) were produced according to the manufacturer's protocol. Ad GFP used as a control was purchased from Qbiogene (Carlsbad, CA).

[0125] Generation of recombinant expression vector carrying FDXR cDNA - full-length wild-type ferredoxin reductase was amplified from human brain cDNA (Clontech, Palo Alto, CA) using the following primers: 5'-CTgTTCCCAgCCATggCTTCgCgCTg-3' (forwa...

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Abstract

Provided herein are methods and compositions for the diagnosis, prognosis and treatment of a cancer associated disorder using the Fhit gene.

Description

[0001] Cross-references to related applications and statements about funded research [0002] This application claims the benefit of Provisional Patent Application Serial No. 60 / 000,480, filed October 26, 2007. This invention was made with Government support under NCI grant numbers CA77738 and CA78890. The government has certain rights in this invention. Background of the invention [0003] The FHIT gene includes the most active common fragile site on chromosome 3p14.2 (1, 2). Fhit expression is lost or reduced in the majority of most types of human tumors due to allelic loss, genomic rearrangement, promoter hypermethylation, or a combination thereof (3, 4). Fhit knockout mice show increased susceptibility to cancer development (5, 6), and FHIT gene therapy prevents tumors in Fhit-deficient mice exposed to carcinogens (7, 8). Fhit restoration by stable transfection in cancer cells has little effect in vitro unless cells are exposed to stress, including environmental stress ...

Claims

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Application Information

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IPC IPC(8): C12N15/00A61K33/243
CPCA61K38/17C12N15/86C12N2710/10343G01N33/5011C12Q1/6886C12Q2600/106G01N2333/90293C12Q2600/136A61K38/46C12Y306/01029A61P35/00A61K33/243A61K45/06A61K48/00A61K31/337C12N7/00C12N2710/10043C12N2710/10071G01N33/57484G01N33/577G01N2333/914
Inventor C·M·克罗斯F·特拉帕索
Owner THE OHIO STATE UNIV RES FOUND