Myt1 overexpression mediates resistance to cell cycle and DNA damage checkpoint kinase inhibitors.

Cell cycle checkpoint kinases serve as important therapeutic targets for various cancers. When they are inhibited by small molecules, checkpoint abrogation can induce cell death or further sensitize cancer cells to other genotoxic therapies. Particularly aberrant Cdk1 activation at the G2/M checkpoint by kinase inhibitors causing unscheduled mitotic entry and mitotic arrest was found to lead to DNA damage and cell death selectively in cancer cells. Promising drugs inhibiting kinases like Wee1 (Adavosertib), Wee1+Myt1 (PD166285), ATR (AZD6738) and Chk1 (UCN-01) have been developed, but clinical data has shown variable efficacy for them with poorly understood mechanisms of resistance. Our lab recently identified Myt1 as a predictive biomarker of acquired resistance to the Wee1 kinase inhibitor, Adavosertib. Here, we investigate the role of Myt1 overexpression in promoting resistance to inhibitors (PD166285, UCN-01 and AZD6738) of other kinases regulating cell cycle progression. We demonstrate that Myt1 confers resistance by compensating Cdk1 inhibition in the presence of these different kinase inhibitors. Myt1 overexpression leads to reduced premature mitotic entry and decreased length of mitosis eventually leading to increased survival rates in Adavosertib treated cells. Elevated Myt1 levels also conferred resistance to inhibitors of ATR or Chk1 inhibitor. Our data supports that Myt1 overexpression is a common mechanism by which cancer cells can acquire resistance to a variety of drugs entering the clinic that aim to induce mitotic catastrophe by abrogating the G2/M checkpoint.

Cell Synchronization Techniques for Studying Mitosis.

Cell synchronization allows the examination of cell cycle progression. Nocodazole and other microtubule poisons have been used extensively to interfere with microtubule function and arrest cells in mitosis. Since microtubules are important for many cellular functions, alternative cell cycle synchronization techniques independent of microtubule inhibition are also used for synchronizing cells in mitosis. Here we describe using nocodazole, STLC, and combining thymidine block with MG132 to synchronize cells in mitosis. These inhibitors are reversible and mitotic cells can be released into the G1 phase synchronously. These techniques can be applied to both Western blot and timelapse imaging to study mitotic progression.

Sub-acute effects of MDMA (+/-3,4-methylenedioxymethamphetamine, "ecstasy") on mood: evidence of gender differences.

RATIONALE: Research with animals suggests that central 5-hydroxytryptamine (5-HT) function may be attenuated for a period following a single dose of +/-3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy'). If the same is true in humans, then functions thought to be modulated by 5-HT may differ in MDMA users compared with non-users a few days after the drug is taken. AIMS: The present study therefore investigated both acute and sub-acute effects of MDMA on mood of recreational users. A second aim was to determine whether these effects differ for females and males. DESIGN: A parallel group design was used to compare 40 participants who reported taking MDMA with 40 participants who reported using illicit substances excluding MDMA (polydrug controls). Participants were assessed on the night of drug use (day 0) and again 4 days later. RESULTS: Female MDMA users showed higher depression scores mid-week than male users or male or female controls. Mid-week depression in female users was correlated with the amount of MDMA taken on day 0. MDMA users rated lower levels of aggression than controls on the night of drug use but significantly higher levels of aggression mid-week, and in males change in aggression correlated with the amount of MDMA taken on the weekend. There was no association between mood and measures of long-term use of MDMA (e.g. years of use). CONCLUSION: Women are more susceptible than men to mid-week low mood following weekend use of MDMA; however, both men and women show increased self-rated aggression. These results are interpreted in terms of an attenuation of 5-HT function for a period following acute use of MDMA.