Involvement of the Fanconi's anemia protein FAC in a pathway that signals to the cyclin B/cdc2 kinase.

Lymphoblastoid cell lines derived from patients with the chromosomal instability disorder Fanconi's anemia (FA) are hyperresponsive to G2 delay and apoptosis induced by cross-linking agents such as mitomycin C (MMC). Here, we investigated whether the protein defective in FA complementation group C (FA-C) cells functions in a pathway that signals to the cdc2 kinase complex, which controls mitotic progression. FA-C lymphoblasts treated with a low dose of MMC (1-5 microM, 1 h) exhibited a protracted G2-M arrest and subsequent apoptosis by 2 days after treatment. This G2-M arrest was mediated by persistent inactivation of the cyclin B1/cdc2 kinase complex characterized by both sustained accumulation of cyclin B1 and tyrosine phosphorylation of cdc2. In phenotypically corrected (wild-type) cells, the same treatment induced only temporal G2-M arrest, associated with a transient inactivation of the cyclin B1/cdc2 kinase complex, after which cells resumed cycling. Treatment with higher dosages (15-30 microM, 1 h) resulted in S-phase arrest and induced a similar high level of apoptosis in FA-C and wild-type cells, accompanied by degradation of cyclin B1 and dephosphorylation of cdc2. In low-dose treated G2-M-arrested FA-C cells, caffeine-dependent activation of cdc2 released the G2-M block but failed to protect against apoptosis, suggesting that apoptosis was not a direct consequence of persistent cdc2 kinase inactivation. Thus, at low doses of MMC, FA-C cells exhibit a unique cyclin B1/cdc2 response that is not observed in wild-type cells treated with an equitoxic high dosage of cross-linker. Although these results do not necessarily implicate a role for FAC in regulating cyclin B/cdc2 kinase activity, available evidence suggests that the FAC protein is involved in a cross-link damage avoidance pathway that signals to this kinase complex.

Hepatoma cells adapted to proliferate under normally lethal hyperthermic stress conditions show rapid decay of thermoresistance and heat shock protein synthesis when returned to 37 degrees C.

H35 hepatoma cultures were adapted to sustained growth at 41.3 degrees C. In these variant cells the 'basic' levels of various heat shock proteins (hsps), especially those of hsp60, 70 and 100, are significantly raised. These cells exhibit a thermoresistance comparable with the induced thermotolerance in normal hepatoma cells heat shocked at 42.5 degrees C for 30 min. However, this resistance of variant cells shows a rapid, exponential decay with a half-time of 2.2 h when the temperature is lowered to 37 degrees C, with a concomitant decrease of the synthesis of hsp60 and 70. Heat shock experiments with variant cells grown at 41.3 degrees C lead to increased thermoresistance and synthesis of hsps when further incubation was performed at the original temperature but not at 37 degrees C. In the latter case, only a 3-h delay in the onset of decay of thermoresistance is observed. However, when the variant cells were incubated at 37 degrees C prior to heat stress normal induction of thermoresistance and hsp synthesis return inversely proportional to the progression of thermoresistance decay. Thermoresistant cells thus seem to be valuable tools in the study of the down-regulation of thermoresistance as well as of hsp synthesis.