Increases in type 1 phosphatase (PP1) activity have been observed in end stage human heart failure, but the role of this enzyme in cardiac function is unknown. To elucidate the functional significance of increased PP1 activity, we generated models with (i) overexpression of the catalytic subunit of PP1 in murine hearts and (ii) ablation of the PP1-specific inhibitor. Overexpression of PP1 (threefold) was associated with depressed cardiac function, dilated cardiomyopathy, and premature mortality, consistent with heart failure. Ablation of the inhibitor was associated with moderate increases in PP1 activity (23%) and impaired ␤-adrenergic contractile responses. Extension of these findings to human heart failure indicated that the increased PP1 activity may be partially due to dephosphorylation or inactivation of its inhibitor. Indeed, expression of a constitutively active inhibitor was associated with rescue of ␤-adrenergic responsiveness in failing human myocytes. Thus, PP1 is an important regulator of cardiac function, and inhibition of its activity may represent a novel therapeutic target in heart failure.

The regulatory-targeting subunit (R GL , also called G M ) of the muscle-specific glycogen-associated protein phosphatase PP1G targets the enzyme to glycogen where it modulates the activity of glycogen-metabolizing enzymes. PP1G/R GL has been postulated to play a central role in epinephrine and insulin control of glycogen metabolism via phosphorylation of R GL . To investigate the function of the phosphatase, R GL knockout mice were generated. Animals lacking R GL show no obvious defects. The R GL protein is absent from the skeletal and cardiac muscle of null mutants and present at ϳ50% of the wild-type level in heterozygotes. Both the level and activity of C1 protein are also decreased by ϳ50% in the R GL -deficient mice. In skeletal muscle, the glycogen synthase (GS) activity ratio in the absence and presence of glucose-6-phosphate is reduced from 0.3 in the wild type to 0.1 in the null mutant R GL mice, whereas the phosphorylase activity ratio in the absence and presence of AMP is increased from 0.4 to 0.7. Glycogen accumulation is decreased by ϳ90%. Despite impaired glycogen accumulation in muscle, the animals remain normoglycemic. Glucose tolerance and insulin responsiveness are identical in wild-type and knockout mice, as are basal and insulin-stimulated glucose uptakes in skeletal muscle. Most importantly, insulin activated GS in both wild-type and R GL null mutant mice and stimulated a GS-specific protein phosphatase in both groups. These results demonstrate that R GL is genetically linked to glycogen metabolism, since its loss decreases PP1 and basal GS activities and glycogen accumulation. However, PP1G/R GL is not required for insulin activation of GS in skeletal muscle, and rather another GS-specific phosphatase appears to be involved.In recent years, the generality that the activity of the type 1 serine/threonine protein phosphatases (PP1) is dictated by the associated noncatalytic subunits has emerged. These ancillary proteins are thought to target the catalytic component (C1) to distinct subcellular locales in proximity to substrates, to confer specificity, and to regulate activity (10,21,33,41). To date, more than 30 C1-binding polypeptides have been identified that direct the enzyme to a variety of subcellular structures, including glycogen (6,24,25,49,59,60), myosin (2), ribosomes (31), nuclei (4, 13), and neuronal structures (5). A subset of C1-binding proteins includes inhibitory proteins such as inhibitors 1 and 2 (48, 67) and DARPP-32 (46). Four C1-glycogen-targeting subunits are presently known. R GL , also called G M , was the first glycogen-binding subunit of PP1 identified (59), and the corresponding holoenzyme, PP1G/ R GL , consists of the 124-kDa R GL protein (60) in association with C1. R GL is exclusively expressed in skeletal and cardiac muscle (37, 60). The NH 2 -terminal 240 amino acids contain binding sites for glycogen and C1 (64), whereas a hydrophobic region between residues 1063 and 1097 in the COOH terminus anchors the protein to membrane (45,60). Of the other three glycoge...

Persistent infection with high-risk human papillomaviruses (HPVs), especially type 16 has been undeniably linked to cervical cancer. The Asian-American (AA) variant of HPV16 is more common in the Americas than the prototype in cervical cancer. The different prevalence is based on three amino acid changes within the E6 protein denoted Q14H/H78Y/L83V. To investigate the mechanism(s) behind this observation, both E6 proteins, in the presence of E7, were evaluated for their ability to extend the life span of and transform primary human foreskin keratinocytes (PHFKs). Longterm cell culture studies resulted in death at passage 9 of vector-transduced PHFKs (negative control), but survival of both E6 PHFKs to passage 65 (and beyond). Compared with E6/E7 PHFKs, AA/E7 PHFKs were significantly faster dividing, developed larger cells in monolayer cultures, showed double the epithelial thickness and expressed cytokeratin 10 when grown as organotypic raft cultures. Telomerase activation and p53 inactivation, two hallmarks of immortalization, were not significantly different between the two populations. Both were resistant to anoikis at later passages, but only AA/E7 PHFKs acquired the capacity for in vitro transformation. Proteomic analysis revealed markedly different protein patterns between E6/E7 and AA/E7, particularly with respect to key cellular metabolic enzymes. Our results provide new insights into the reasons underlying the greater prevalence of the AA variant in cervical cancer as evidenced by characteristics associated with higher oncogenic potential.

IntroductionThe taxanes paclitaxel and docetaxel are widely used in the treatment of breast, ovarian, and other cancers. Although their cytotoxicity has been attributed to cell-cycle arrest through stabilization of microtubules, the mechanisms by which tumor cells die remains unclear. Paclitaxel has been shown to induce soluble tumor necrosis factor alpha (sTNF-α) production in macrophages, but the involvement of TNF production in taxane cytotoxicity or resistance in tumor cells has not been established. Our study aimed to correlate alterations in the TNF pathway with taxane cytotoxicity and the acquisition of taxane resistance.MethodsMCF-7 cells or isogenic drug-resistant variants (developed by selection for surviving cells in increasing concentrations of paclitaxel or docetaxel) were assessed for sTNF-α production in the absence or presence of taxanes by enzyme-linked immunosorbent assay (ELISA) and for sensitivity to docetaxel or sTNF-α by using a clonogenic assay (in the absence or presence of TNFR1 or TNFR2 neutralizing antibodies). Nuclear factor (NF)-κB activity was also measured with ELISA, whereas gene-expression changes associated with docetaxel resistance in MCF-7 and A2780 cells were determined with microarray analysis and quantitative reverse transcription polymerase chain reaction (RTqPCR).ResultsMCF-7 and A2780 cells increased production of sTNF-α in the presence of taxanes, whereas docetaxel-resistant variants of MCF-7 produced high levels of sTNF-α, although only within a particular drug-concentration threshold (between 3 and 45 nM). Increased production of sTNF-α was NF-κB dependent and correlated with decreased sensitivity to sTNF-α, decreased levels of TNFR1, and increased survival through TNFR2 and NF-κB activation. The NF-κB inhibitor SN-50 reestablished sensitivity to docetaxel in docetaxel-resistant MCF-7 cells. Gene-expression analysis of wild-type and docetaxel-resistant MCF-7, MDA-MB-231, and A2780 cells identified changes in the expression of TNF-α-related genes consistent with reduced TNF-induced cytotoxicity and activation of NF-κB survival pathways.ConclusionsWe report for the first time that taxanes can promote dose-dependent sTNF-α production in tumor cells at clinically relevant concentrations, which can contribute to their cytotoxicity. Defects in the TNF cytotoxicity pathway or activation of TNF-dependent NF-κB survival genes may, in contrast, contribute to taxane resistance in tumor cells. These findings may be of strong clinical significance.