Susan L. Mooberry

Induction of apoptosis by cryptophycin 1, a new antimicrotubule agent

The ability of cryptophycin 1, a new potent cytotoxic antimicrotubule agent, to initiate apoptosis was studied. Treatment of cells with cryptophycin 1 (50 pM) rapidly caused morphological changes consistent with the induction of apoptosis. DNA strand breakage and fragmentation of the DNA into oligonucleosome‐sized fragments was observed, and this coincided with the loss of cellular DNA. Activation of the cysteine protease CPP32 (caspase 3, YAMA, apopain), a member of the ICE/CED‐3‐like protease family of apoptosis effectors, was consistent with the execution of cell death by a coordinated sequence of events. Low concentrations of cryptophycin 1 caused mitotic arrest with the formation of abnormal mitotic spindles without affecting interphase microtubule structures. Unlike other microtubule active agents, cryptophycin‐induced mitotic arrest persisted for only a brief period before the onset of apoptosis. There was no evidence of release from G2/M cell cycle arrest. Our results show that low concentrations of cryptophycin 1 (50 pM) initiated cell death consistent with apoptosis. These data suggest that the cytotoxic effects of cryptophycin 1 are due in part to its ability to initiate apoptosis rapidly. Int. J. Cancer 73:440–448, 1997.

Cryptophycin 1 binds to tubulin at a site distinct from the colchicine binding site and at a site that may overlap the vinca binding site

Cryptophycin 1 is a new cytotoxic antimicrotubule agent with excellent antitumor activity. The methods of Sackett (Biochemistry, 34, 7010-7019, 1995), utilizing the selective and specific proteolysis of alpha- and beta-tubulin by trypsin and chymotrypsin, was used to identify the cryptophycin 1 binding site on tubulin. Occupancy of the colchicine or vinca binding sites causes changes in the structure of tubulin that can be detected by proteolysis with trypsin and chymotrypsin. The addition of cryptophycin 1 to tubulin causes changes in both the tryptic and chymotryptic cleavage of tubulin consistent with occupation of the vinca binding site and distinct from occupation of the colchicine binding site. The effects of cryptophycin 1 on the tryptic digests are identical to the effects seen with vinblastine and differ saliently from the effects of maytansine and rhizoxin, other agents known to bind to the vinca site. The data suggest that the binding site of cryptophycin 1 may overlap the vinca binding site on tubulin.

Tubercidin stabilizes microtubules against vinblastine-induced depolymerization, a taxol-like effect

A sensitive assay for the detection of microtubule-stabilizing agents [1] was used to screen an extensive collection of cyanobacterial and microalgal extracts. The hydrophilic extract of the cyanobacterium, Plectonema radiosum (UH isolate IC-70-1), exhibited microtubule-stabilizing activity. Bioassay-directed purification of the active compound yielded tubercidin (7-deazaadenosine), a potent cytotoxic nucleoside analog. Further studies revealed that tubercidin protected a population of cellular microtubules against vinblastine-induced depolymerization, a microtubule-stabilizing, taxol-like effect. The microtubule-stabilizing effect of tubercidin is dose dependent and limited by the cytotoxicity of the agent. Tubercidin represents another natural product that interacts with microtubules and is one of the few to cause microtubule stabilization.

A sensitive assay for taxol and other microtubule-stabilizing agents

The ability of taxol to protect microtubules in cultured human ovarian carcinoma cells from drug- and cold-induced depolymerization was characterized as a functional assay for microtubule stabilizing agents. Treatment of the cells with concentrations of vinblastine or colchicine of 50 nM or greater, or incubation at 4 degrees C resulted in complete depolymerization of cytoplasmic microtubules. Pretreatment with taxol for 3 h enabled the cells to maintain substantial numbers of microtubules following the application of vinblastine or colchicine. This protective effect was easily observed at 50 nM taxol, whereas taxol-induced microtubule bundling was observed only at concentrations of 500 nM or greater. Concentrations of taxol as low as 10 nM stabilized microtubules against cold-induced depolymerization. Therefore, protection of microtubules from drug- and cold-induced depolymerization provides a sensitive functional assay for taxol. These systems should be similarly effective in identifying novel compounds which stabilize microtubules.

Monoclonal antibodies to taxanes that neutralize the biological activity of paclitaxel

Recently it has been proposed that drug-specific neutralizing antibodies may limit side-effects that occur during chemotherapy. These studies were undertaken to determine if monoclonal antibodies, 3C6 specific to paclitaxel and 8A10 specific to taxane diterpenes, are inhibitors of paclitaxel-induced inhibition of proliferation and cellular microtubule and nuclear changes. The results show that 3C6 and 8A10 each inhibit paclitaxel-induced cytotoxicity, microtubular bundling, stabilization from vinblastine-induced microtubule depolymerization and the formation of micronuclei. We conclude that these antibodies effectively neutralize paclitaxel activity in vitro and that they may be useful to determine if antibody-blocking strategies can prevent dose-limiting toxicities.