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Methods and Compositions for the Diagnosis of Cancer Susceptibilities and Defective DNA Repair Mechanisms and Treatment Thereof

a technology of dna repair and cancer susceptibility, applied in the direction of immunoglobulins against animals/humans, biological material analysis, peptides, etc., can solve the problems of complex diagnosis of fa, inability to differentiate existing diagnostic tests that cannot distinguish fa carriers from the general population, so as to reduce intracellular drug levels, increase drug inactivation, and reduce inward transport

Inactive Publication Date: 2011-10-20
OREGON HEALTH & SCI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0042]In an embodiment of the invention, a method is provided for determining a therapeutic protocol for a subject having a cancer, that includes (a) determining if a deficiency in FANCD2-L occurs in a cell sample from the subject by measuring FANCD2 isoforms using specific antibodies; (b) if a deficiency is detected in (a), then determining whether the deficiency is a result of genetic defect in non-cancer cells; and (c) if (b) is positive, reducing the use of a therapeutic protocol that causes increased DNA damage so as to protect normal tissue in the subject and if (b) is negative, and the deficiency is contained within a genetic defect in cancer cells only, then increasing the use of a therapeutic protocol that causes increased DNA damage so as to adversely affect the cancer cells.
[0077]As used herein, the term “cancer therapeutic” refers to a compound that prevents the onset or progression of cancer or prevents cancer metastasis or reduces, delays, or eliminates the symptoms of cancer.
[0081]As used herein, “resistance to one or more anti-neoplastic agents” refers the ability of cancer cells to develop resistance to anticancer drugs. Mechanisms of drug resistance include decreased intracellular drug levels caused by an increased drug efflux or decreased inward transport, increased drug inactivation, decreased conversion of drug to an active form, altered amount of target enzyme or receptor (gene amplification), decreased affinity of target enzyme or receptor for drug, enhanced repair of the drug-induced defect, decreased activity of an enzyme required for the killing effect (topoisomerase II). In a preferred embodiment of the invention, drug resistance refers to the enhanced repair of DNA damage induced by one or more anti-neoplastic agents. In another preferred embodiment of the invention, the enhanced repair of DNA damage induced by one or more anti-neoplastic agents is due to a constitutively active Fanconi Anemia / BRCA DNA repair pathway.

Problems solved by technology

Diagnosis of FA is complicated by the wide variability in FA patient phenotype.
Moreover, existing diagnostic tests do not differentiate FA carriers from the general population.
Diagnosing cancer susceptibility is complicated because of the large number of regulatory genes and biochemical pathways that have been implicated in the formation of cancers.
Genetic lesions that are associated with defective repair mechanisms may give rise to defective cell division and apoptosis which in turn may increase a patient's susceptibility to cancer.

Method used

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  • Methods and Compositions for the Diagnosis of Cancer Susceptibilities and Defective DNA Repair Mechanisms and Treatment Thereof
  • Methods and Compositions for the Diagnosis of Cancer Susceptibilities and Defective DNA Repair Mechanisms and Treatment Thereof
  • Methods and Compositions for the Diagnosis of Cancer Susceptibilities and Defective DNA Repair Mechanisms and Treatment Thereof

Examples

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example 1

Experimental Protocols Used in Examples 2-8

[0239]Cell Lines and Culture Conditions. Epstein-Barr virus (EBV) transformed lymphoblasts were maintained in RPMI media supplemented with 15% heat-inactivated fetal calf serum (FCS) and grown in a humidified 5% CO2-containing atmosphere at 37° C. A control lymphoblast line (PD7) and FA lymphoblast lines (FA-A (HSC72), FA-C (PD-4), FA-D (PD-20), FA-F (EUFA121), and FA-G (EUFA316) have been previously described (de Winter et al., Nat. Genet., (1998) Vol. 20, pp. 281-283) (Whitney et al., Nat. Genet., (1995) Vol. 11, pp. 341-343) (Yamashita et al., P.N.A.S., (1994) Vol. 91, pp. 6712-6716) (de Winter et al., Am. J. Hum. Genet., (2001), Vol. 57, pp. 1306-1308). PD81 is a lymphoblast cell line from an FA-A patient. The SV40-transformed FA fibroblasts, GM6914, PD426, FAG326SV and PD20F, as well as HeLa cells, were grown in DMEM supplemented with 15% FCS. FA cells (both lymphoblasts and fibroblasts) were functionally complemented with pMMP retrovi...

example 2

The FA Genes Interact in a Common Cellular Pathway

[0251]Normal lymphoblasts express two isoforms of the FANCD2 protein, a short form (FANCD2-S, 155 kD) and a long form (FANCD2-L, 162 kD). FIG. 1 shows what happened when whole cell extracts were prepared from a lymphoblast line and cellular proteins were immunoprecipitated with an anti-FANCD2 antiserum. Normal wild type cells expressed two isoforms of the FANCD2 protein—a low molecular weight isoform FANCD2-S (155 kD isoform) and a high molecular weight isoform (FANCD2-L) (162 kD isoform). FANCD2-S is the primary translation product of the cloned FANCD2 cDNA. We next evaluated a large series of FA lymphoblasts and fibroblasts for expression of the FANCD2 isoforms (Table 5). Correction of these FA cell lines with the corresponding FA cDNA resulted in functional complementation and restoration of the high molecular weight isoform, FANCD2-L.

[0252]As previously described, FA cells are sensitive to the DNA crosslinking agent, MMC, and in ...

example 3

The FA Protein Complex is Required for the Monoubiquitination of FANCD2

[0253]The high molecular weight isoform of FANCD2 could result from one or more mechanisms, including alternative splicing of the FANCD2 mRNA or post-translational modification(s) of the FANCD2 protein. Treatment with phosphatase did not convert FANCD2-L to FANCD2-S, demonstrating that phosphorylation alone does not account for the observed difference in their molecular mass.

[0254]In order to identify other possible post-translational modifications of FANCD2, we initially sought cellular conditions which regulate the conversion of FANCD2-S to FANCD2-L (FIGS. 1B, C). Since FA cells are sensitive to MMC and IR, we reasoned that these agents might regulate the conversion of FANCD2-S to FANCD2-L in normal cells. Interestingly, HeLa cells treated with MMC (FIG. 1B, lanes 1-6) or IR (FIG. 1C, lanes 1-6) demonstrated a dose-dependent increase in the expression of the FANCD2-L isoform.

[0255]To determine whether FANCD2-L ...

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Abstract

Methods and compositions for the diagnosis of cancer susceptibilities, defective DNA repair mechanisms and treatments thereof are provided. Among sequences provided here, the FANCD2 gene has been identified, and probes and primers are provided for screening patients in genetic-based tests and for diagnosing Fanconi Anemia and cancer. The FANCD2 gene can be targeted in vivo for preparing experimental mouse models for use in screening new therapeutic agents for treating conditions involving defective DNA repair. The FANCD2 polypeptide has been sequenced and has been shown to exist in two isoforms identified as FANCD2-S and the monoubiquinated FANCD-L form. Antibodies including polyclonal and monoclonal antibodies have been prepared that distinguish the two isoforms and have been used in diagnostic tests to determine whether a subject has an intact Fanconi Anemia / BRCA pathway.

Description

RELATED APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 10 / 165,099, filed Jun. 6, 2002, which is a continuation-in-part of U.S. application Ser. No. 09 / 998,027, filed Nov. 2, 2001, which in turn claims priority from provisional application 60 / 245,756, filed Nov. 3, 2000. The entire contents of each of the above applications are incorporated herein by reference.GOVERNMENT SUPPORT[0002]The work described herein was supported by the National Institute of Health, NIH Grant No. Health grants RO1HL52725-04, RO1DK43889-09, 1PO1HL48546, and PO1HL54785-04. The US Government has certain rights to the claimed invention.BACKGROUND[0003]The present invention relates to the diagnosis of cancer susceptibilities in subjects having a defect in the FANCD2 gene and the determination of suitable treatment protocols for those subjects who have developed cancer. Animal models with defects in the FANCD2 gene can be used to screen for therapeutic agents.[0004]Fanconi Anemi...

Claims

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Application Information

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IPC IPC(8): C40B30/06G01N33/566C12N5/071C07K14/47C12Q1/02C07K16/18C12Q1/68G01N33/50G01N33/574
CPCA01K2217/05A01K2217/075C07K14/47C07K16/18C12Q1/6886C12Q2600/156G01N33/6893G01N33/5091G01N33/574G01N33/57484G01N2500/00C12Q1/025C12Q2600/158G01N33/5011G01N2333/47G01N2800/52
Inventor D'ANDREA, ALAN D.TANIGUCHI, TOSHIYASUTIMMERS, CYNTHIAGROMPE, MARKUSFOX, EDWARD A.
Owner OREGON HEALTH & SCI UNIV
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