Each mouse received three intraperitoneal implants (one of each tumour cell line) and three subcutaneous implants (one of each tumour cell line as in intraperitoneal implants). For each experiment, three mice/dose were used. Vehicle controls consisted of six mice that received only the vehicle, 10% (v/v) DMSO in saline/0.05% Tween 80 (Sigma, St. Louis, MO, U.S.A.). Evaluation of 12 tumour cell lines required a total of four experiments, each containing three tumour cell lines. Tuba (Tubulysin A) was administered at two doses, 0.04 and 0.06 mg/kg, via the intraperitoneal route once daily for 4 days. Twenty four hours after the last treatment, the fibreswere collected, assessed for viable cell mass, using a stable endpoint MTT (2,3-bis-(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide, disodium salt) cell viability assay, and percentage net cell growth and the scores were calculated by comparing with the zero time viable cell mass

The antiproliferative activity of tuba (Tubulysin A) in a panel of 60 human cancer cell lines was performed at the NCI using a standard SRB (sulphorhodamine B) assay. TubA was tested at five serial 1 log dilutions (concentrations 0.01–100 μM) using the standard 48 h incubation.