Mark W. Wagner

Calcineurin Is Activated in Rat Hearts With Physiological Left Ventricular Hypertrophy Induced by Voluntary Exercise Training

BACKGROUND Calcineurin may play a pivotal role in the signaling of cardiac hypertrophy; since this hypothesis was first put forward, controversial reports have been published using various experimental models. This study was designed to compare the physiological left ventricular hypertrophy (LVH) induced by voluntary exercise with LVH induced by aortic constriction and to determine whether calcineurin participates in the signaling of exercise-induced LVH. METHODS AND RESULTS Wistar rats were assigned to 1 of the following 5 groups: 10 weeks of voluntary exercise (EX), a sedentary regimen, a 1-week (AC1) or 4-week (AC4) ascending aortic constriction period, or a sham operation. EX rats ran 2.4+/-0.7 km/day voluntarily in specially manufactured cages; this was associated with an increase of LV diastolic dimension and stroke volume. Myocardial calcineurin activity markedly increased in EX rats (46.4+/-8.3 versus 18.4+/-0.5 pmol. min(-1). mg(-1) in sedentary rats; P<0.001) and in AC1 rats (44.9+/-6.7 versus 22.1+/-3.7 pmol. min(-1). mg(-1) in sham-operated rats; P<0.001), but not in AC4 rats (29.0+/-3.4 pmol. min(-1). mg(-1)). Treatment with cyclosporin A completely inhibited the development of LVH in EX rats, but it only partially attenuated the development of LVH in AC4 rats. CONCLUSIONS Calcineurin was activated in exercise-induced physiological LVH and in the developing phase of LVH (AC1), but not in decompensated pressure-overload hypertrophy (AC4). Cyclosporin therapy for the prevention of LVH may be harmful because it does not block the development of pathological hypertrophy but rather that of favorable adaptive hypertrophy.

Akt1/PKB upregulation leads to vascular smooth muscle cell hypertrophy and polyploidization

Vascular smooth muscle cells (VSMCs) at capacitance arteries of hypertensive individuals and animals undergo marked age- and blood pressure-dependent polyploidization and hypertrophy. We show here that VSMCs at capacitance arteries of rat models of hypertension display high levels of Akt1/PKB protein and activity. Gene transfer of Akt1 to VSMCs isolated from a normotensive rat strain was sufficient to abrogate the activity of the mitotic spindle cell-cycle checkpoint, promoting polyploidization and hypertrophy. Furthermore, the hypertrophic agent angiotensin II induced VSMC polyploidization in an Akt1-dependent manner. These results demonstrate that Akt1 regulates ploidy levels in VSMCs and contributes to vascular smooth muscle polyploidization and hypertrophy during hypertension.

A role for DNA mismatch repair in sensing and responding to fluoropyrimidine damage

The phenomenon of damage tolerance, whereby cells incur DNA lesions that are nonlethal, largely ignored, but highly mutagenic, appears to play a key role in carcinogenesis. Typically, these lesions are generated by alkylation of DNA or incorporation of base analogues. This tolerance is usually a result of the loss of specific DNA repair processes, most often DNA mismatch repair (MMR). The availability of genetically matched MMR-deficient and -corrected cell systems allows dissection of the consequences of this unrepaired damage in carcinogenesis as well as the elucidation of cell cycle checkpoint responses and cell death consequences. Recent data indicate that MMR plays an important role in detecting damage caused by fluorinated pyrimidines (FPs) and represents a repair system that is probably not the primary system for detecting damage caused by these agents, but may be an important system for correcting key mutagenic lesions that could initiate carcinogenesis. In fact, clinical studies have shown that there is no benefit of FP-based adjuvant chemotherapy in colon cancer patients exhibiting microsatellite instability, a hallmark of MMR deficiency. MMR-mediated damage tolerance and futile cycle repair processes are discussed, as well as possible strategies using FPs to exploit these systems for improved anticancer therapy.

DNA Mismatch Repair-dependent Activation of c-Abl/p73α/GADD45α-mediated Apoptosis

Cells with functional DNA mismatch repair (MMR) stimulate G2 cell cycle checkpoint arrest and apoptosis in response to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). MMR-deficient cells fail to detect MNNG-induced DNA damage, resulting in the survival of “mutator” cells. The retrograde (nucleus-to-cytoplasm) signaling that initiates MMR-dependent G2 arrest and cell death remains undefined. Since MMR-dependent phosphorylation and stabilization of p53 were noted, we investigated its role(s) in G2 arrest and apoptosis. Loss of p53 function by E6 expression, dominant-negative p53, or stable p53 knockdown failed to prevent MMR-dependent G2 arrest, apoptosis, or lethality. MMR-dependent c-Abl-mediated p73α and GADD45α protein up-regulation after MNNG exposure prompted us to examine c-Abl/p73α/GADD45α signaling in cell death responses. STI571 (Gleevec™, a c-Abl tyrosine kinase inhibitor) and stable c-Abl, p73α, and GADD45α knockdown prevented MMR-dependent apoptosis. Interestingly, stable p73α knockdown blocked MMR-dependent apoptosis, but not G2 arrest, thereby uncoupling G2 arrest from lethality. Thus, MMR-dependent intrinsic apoptosis is p53-independent, but stimulated by hMLH1/c-Abl/p73α/GADD45α retrograde signaling.

Role of c-Abl Kinase in DNA Mismatch Repair-dependent G2 Cell Cycle Checkpoint Arrest Responses

Current published data suggest that DNA mismatch repair (MMR) triggers prolonged G2 cell cycle checkpoint arrest after alkylation damage from N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) by activating ATR (ataxia telangiectasia-Rad3-related kinase). However, analyses of isogenic MMR-proficient and MMR-deficient human RKO colon cancer cells revealed that although ATR/Chk1 signaling controlled G2 arrest in MMR-deficient cells, ATR/Chk1 activation was not involved in MMR-dependent G2 arrest. Instead, we discovered that disrupting c-Abl activity using STI571 (Gleevec™, a c-Abl inhibitor) or stable c-Abl knockdown abolished MMR-dependent p73α stabilization, induction of GADD45α protein expression, and G2 arrest. In addition, inhibition of c-Abl also increased the survival of MNNG-exposed MMR-proficient cells to a level comparable with MMR-deficient cells. Furthermore, knocking down GADD45α (but not p73α) protein levels affected MMR-dependent G2 arrest responses. Thus, MMR-dependent G2 arrest responses triggered by MNNG are dependent on a human MLH1/c-Abl/GADD45α signaling pathway and activity. Furthermore, our data suggest that caution should be taken with therapies targeting c-Abl kinase because increased survival of mutator phenotypes may be an unwanted consequence.

Ectopic Expression of cdc2/cdc28 Kinase Subunit Homo sapiens 1 Uncouples Cyclin B Metabolism from the Mitotic Spindle Cell Cycle Checkpoint

ABSTRACT Primary human fibroblasts arrest growth in response to the inhibition of mitosis by mitotic spindle-depolymerizing drugs. We show that the mechanism of mitotic arrest is transient and implicates a decrease in the expression of cdc2/cdc28 kinase subunit Homo sapiens 1 (CKsHs1) and a delay in the metabolism of cyclin B. Primary human fibroblasts infected with a retroviral vector that drives the expression of a mutant p53 protein failed to downregulate CKsHs1 expression, degraded cyclin B despite the absence of chromosomal segregation, and underwent DNA endoreduplication. In addition, ectopic expression of CKsHs1 interfered with the control of cyclin B metabolism by the mitotic spindle cell cycle checkpoint and resulted in a higher tendency to undergo DNA endoreduplication. These results demonstrate that an altered regulation of CKsHs1 and cyclin B in cells that carry mutant p53 undermines the mitotic spindle cell cycle checkpoint and facilitates the development of aneuploidy. These data may contribute to the understanding of the origin of heteroploidy in mutant p53 cells.