Kitagawa K: Human project



17-AAG, the Hsp90 inhibitor

The Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG), which is currently in clinical trials, is thought to exert antitumor activity by simultaneously targeting several oncogenic signaling pathways. We have found a novel mechanism by which 17-AAG inhibits cell proliferation and provide the first evidence of HSP90 being required for assembly of kinetochore protein complexes in humans. 17-AAG causes localization of several kinetochore proteins (including CENP-I and CENP-H) but not CENP-B and CENP-C (Fig. 3). It also induces a spindle checkpoint–dependent mitotic arrest and chromosome misalignment and aneuploidy. Also, HSP90 associates with SGT1 (suppressor of G2 allele of skp1; SUGT1) in human cells and depletion of SGT1 sensitizes HeLa cells to 17-AAG. Overexpression of SGT1 restores the localization of specific kinetochore proteins and chromosome alignment in cells treated with 17-AAG. Biochemical and genetic results suggest that HSP90, by interacting with SGT1 (SUGT1), is required for kinetochore assembly. Time-course experiments have revealed that transient treatment with 17-AAG between late S andG2/M phases causes substantial delocalization of CENP-H and CENP-I,  strongly suggesting that HSP90 participates in kinetochore assembly in a cell cycle–dependent manner.


 

Fig 3. Delocalization of kinetochore proteins in response to 17-AAG treatment.  


CENP-H (a central kinetochore protein) signals at the kinetochore were diminished during prophase, metaphase, and interphase after treatment with 17-AAG. CREST, an anti-centromere antibody that originated from patients with CREST, detects mainly CENP-A (the centromeric histone H3), CENP-B (an inner heterochromatin kinetochore protein), and CENP-C (an inner/central plate kinetochore protein).


Caspase-Independent Mitotic Death (CIMD)

Although some evidence suggests that apoptosis occurs during mitosis, the relationship between apoptosis or programmed cell death during mitosis and the spindle checkpoint is unknown. We have recently identified a novel type of mitotic cell death, which we term caspase-independent mitotic death (CIMD). In BUB1-deficient (but not MAD2-deficient) cells, CIMD is induced by conditions that activate the spindle checkpoint (i.e., cold shock or treatment with nocodazole, paclitaxel, or 17-AAG). CIMD depends on p73, a homolog of p53, but not on p53. It also depends on the apoptosis-inducing factor (AIF) and endonuclease G (Endo G), which are effectors of caspase-independent cell death. When BUB1 is completely depleted, aneuploidy occurs instead of CIMD. We propose that CIMD can be the cell death mechanism that protects cells from aneuploidy by inducing the death of cells prone to substantial chromosome missegregation. Our study also shows that previous evaluations of the spindle checkpoint activity in mutant or cancer cells by monitoring mitotic index could be misleading.

Fig 4. (A) A model that describes how chromosome loss or nondisjunction occurs in spindle checkpoint–defective cells (MAD2-depleted cells or complete BUB1-depleted cells). In spindle checkpoint–mutant cells, the spindle checkpoint is not activated even if there are defects in kinetochore–microtubule attachment. No mitotic delay occurs, which results in premature exit from mitosis. Thus, substantial chromosome loss or nondisjunction occurs, and presumably cell death follows.


(B) A model that describes the same scenario in partial BUB1-depleted cells (when CIMD occurs). Here, defects in kinetochore–microtubule attachment induce lethal DNA fragmentation. Because cells are still arrested in mitosis, the mitotic index is unchanged. Therefore, the spindle checkpoint appears to be active (ON). But, the cells were dead at that moment in mitosis.


Fig 5. CIMD occurs in BUB1-depleted cells in the presence of microtubule inhibitors or 17-AAG. HeLa cells that are BUB1-depleted and 17-AAG–treated exhibit DNA fragmentation (TUNEL-positive) during mitosis. Forty-eight hours after HeLa cells were transfected with BUB1 siRNA, they were incubated with 17-AAG (+17AAG, 500 nM) for 24 h at 37°C.  Fixed samples were stained by using an in situ cell death detection system that contained TMR red (red), an anti–phosphorylated histone H3 (p-H3) mouse monoclonal antibody, and FITC–conjugated secondary antibodies (green). DNA was stained with DAPI (blue) to visualize metaphase (top) and prometaphase (bottom) cells.

 

Fig 6. Pathway showing how and where the treatments used in our studies act on our proposed model.

The anticancer drug 17-AAG, microtubule inhibitors, and cold shock induce defects in the kinetochore­–microtubule attachment, which lead to CIMD in Bub1-deficient cells.

(#1) This assay identifies inhibitors that are similar to these anticancer agents. (#2) Bub1 inhibitors (that can induce CIMD in mitotically dividing caner cells in combination with 17-AAG and microtubule inhibitors) are likely to synergistically work as anticancer drugs with 17-AAG and microtubule inhibitors.  (#3) An siRNA against the factor that functions downstream of BUB1 inhibits CIMD. siRNA screens will be able to fill up the pathway.

 


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