Detection and modulation of IAPs and NAIP for the diagnosis and treatment of proliferative disease

Abstract

Disclosed are diagnostic and prognostic kits for the detection and treatment of proliferative diseases such as ovarian cancer, breast cancer, and lymphoma. Also disclosed are cancer therapeutics utilizing IAP antisense nucleic acids, IAP fragments, and antibodies which specifically bind IAP polypeptides.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the human xiap cDNA sequence (SEQ ID NO:3) and the XIAP polypeptide sequence (SEQ ID NO:4).

FIG. 2 is the human hiap-1 cDNA sequence (SEQ ID NO:5) and the HIAP-1 polypeptide sequence (SEQ ID NO:6).

FIG. 3 is the human hiap-2 cDNA sequence (SEQ ID NO:7) and the HIAP-2 polypeptide sequence (SEQ ID NO:8). The sequence absent in the hiap-2-A variant is boxed.

FIG. 4 is the murine xiap (also referred to as “miap-3”) cDNA sequence (SEQ ID NO:9) and encoded murine XIAP polypeptide sequence (SEQ ID NO:10).

FIG. 5 is the murine hiap-1 (also referred to as “miap-1”) cDNA sequence (SEQ ID NO:11) and the encoded murine HIAP-1 polypeptide sequence (SEQ ID NO:12).

FIG. 6 is the murine hiap-2 (also referred to as “miap-2”) cDNA sequence (SEQ ID NO:13) and the encoded murine HIAP-2 polypeptide (SEQ ID NO:14).

FIG. 7 is a photograph of a Northern blot illustrating human hiap-1 and hiap-2 mRNA expression in human tissues.

FIG. 8 is a photograph of a Northern blot illustrating human hiap-2 mRNA expression in human tissues.

FIG. 9 is a photograph of a Northern blot illustrating human xiap mRNA expression in human tissues.

FIG. 10A-10D are graphs depicting suppression of apoptosis by XIAP, HIAP-1, HIAP-2, bcl-2, smn, and 6-myc.

FIG. 11 is a photograph of an agarose gel containing cDNA fragments that were amplified, with hiap-1-specific primers, from RNA obtained from Raji, Ramos, EB-3, Burkitt's lymphoma cells, and Jiyoye cells, and cells from normal placenta.

FIG. 12 is a photograph of a Western blot containing protein extracted from Jurkat and astrocytoma cells stained with an anti-XIAP antibody. The position and size of a series of marker proteins is indicated.

FIG. 13 is a photograph of a Western blot containing protein extracted from Jurkat cells following treatment as described in Example XII. The blot was stained with a rabbit polyclonal anti-XIAP antibody. Lane 1, negative control; lane 2, anti-Fas antibody; lane 3, anti-Fas antibody and cycloheximide; lane 4, TNF-α; lane 5, TNF-α and cycloheximide.

FIG. 14 is a photograph of a Western blot containing protein extracted from HeLa cells following exposure to anti-Fas antibodies. The blot was stained with a rabbit polyclonal anti-XIAP antibody. Lane 1, negative control; lane 2, cycloheximide; lane 3, anti-Fas antibody; lane 4, anti-Fas antibody and cycloheximide; lane 5, TNF-α; lane 6, TNF-α and cycloheximide.

FIGS. 15A and 15B are photographs of Western blots stained with rabbit polyclonal anti-XIAP antibody. Protein was extracted from HeLa cells (FIG. 21A) and Jurkat cells (FIG. 21B) immediately, 1, 2, 3, 5, 10, and 22 hours after exposure to anti-Fas antibody.

FIGS. 16A and 16B are photographs of Western blots stained with an anti-CPP32 antibody (FIG. 16A) or a rabbit polyclonal anti-XIAP antibody (FIG. 16B). Protein was extracted from Jurkat cells immediately, 3 hours, or 7 hours after exposure to an anti-Fas antibody. In addition to total protein, cytoplasmic and nuclear extracts are shown.

FIG. 17 is a photograph of a polyacrylamide gel following electrophoresis of the products of an in vitro XIAP cleavage assay.

FIGS. 18 and 19 shows the increased level of HIAP-1 and HIAP-2 mRNA, respectively, in breast cancer cell lines having p53 mutations (lanes 5-7). The bottom portion of the figure shows the control.

FIG. 20 shows the influence of Taxol on DNA fragmentation in Cisplatin-sensitive (right) and resistant (left) human ovarian epithelial cancer cells.

FIG. 21 shows the influence of Cisplatin on DNA fragmentation in sensitive (right) and resistant (left) human ovarian epithelial cancer cells.

FIG. 22 shows the effects of Taxol on XIAP and Hiap-2 protein levels in Cisplatin sensitive (right) and resistant (left) human ovarian epithelial cancer cells.

FIGS. 23A and 23B show the influence of Taxol and TGFβ on HIAP-2 mRNA levels in Cisplatin sensitive (right) and resistant (left) human epithelial cancer cells.

FIGS. 24A and 24B show the effect of TGFβ on XIAP protein expression (FIG. 24A) and DNA fragmentation (FIG. 24B) in Cisplatin sensitive and resistant cells.

Claims

1. An antisense nucleic acid of length sufficient to inhibit an inhibitor of apoptosis (IAP) biological activity in vivo, wherein the antisense nucleic acid is complementary to a mammalian IAP nucleic acid sequence selected from the group consisting of: human X-linked IAP (XIAP) (SEQ ID NO:3), human IAP-1 (HIAP-1) (SEQ ID NO:5), human IAP-2 (HIAP-2) (SEQ ID NO:7), murine XIAP (SEQ ID NO:9), murine XIAP-1 (SEQ ID NO:11) and murine XIAP-2 (SEQ ID NO:13).

2. The antisense nucleic acid according to claim 1 in which the inhibitor of apoptosis (IAP) biological activity is inhibition of apoptosis.

3. The antisense nucleic acid according to claim 1 in which the inhibitor of apoptosis (IAP) biological activity is inhibited by at least 25%.

4. The antisense nucleic acid according to claim 1 in which the inhibitor of apoptosis (IAP) is human IAP-1 (HIAP-1).

5. The antisense nucleic acid according to claim 1 in which the inhibitor of apoptosis (IAP) is human IAP-2 (HIAP-2).

6. The antisense nucleic acid according to claim 1 in which the inhibitor of apoptosis (IAP) is human X-linked IAP (XIAP).

7. The antisense nucleic acid according to claim 1 in which the antisense nucleic acid is murine.

8. The antisense nucleic acid according to claim 1 in which the antisense nucleic acid is human.