Ming-Jer Tang

KCl cotransport is an important modulator of human cervical cancer growth and invasion

Cervical cancer is a major world health problem for women, but the pathophysiology of this disease has received scant attention. Here we show that the growth and invasion of cervical cancer cells are strongly linked the expression and activity of the KCl cotransporter (KCC), an important regulator of the ionic and cellular osmotic homeostasis. Functional assays of KCl cotransport activation by osmotic swelling, staurosporine, and N-ethylmaleimide indicate that removal of the N-terminal 117 amino acids from KCC1 produces a dominant-negative loss-of-function phenotype for KCl cotransport in human cervical cancer cells. The capability for regulatory volume decrease is much attenuated in the loss-of-function KCC mutant cervical cancer cells. The loss-of-function KCC mutant cervical cancer cells exhibit inhibited cell growth accompanied by decreased activity of the cell cycle gene products retinoblastoma and cdc2 kinase. Reduced cellular invasiveness is in parallel by reduced expression of αvβ3 and α6β4 integrins, accompanied by decreased activity of matrix metalloproteinase 2 and 9. Inhibition of tumor growth in SCID mice confirms the crucial role of KCC in promoting cervical cancer growth and invasion. Thus, blockade of KCl cotransport may be a useful therapeutic adjunctive strategy to retard or prevent cervical cancer invasion.

Bcl-2 Rescues Ceramide- and Etoposide-induced Mitochondrial Apoptosis through Blockage of Caspase-2 Activation

Recent studies indicate that caspase-2 is involved in the early stage of apoptosis before mitochondrial damage. Although the activation of caspase-2 has been shown to occur in a large protein complex, the mechanisms of caspase-2 activation remain unclear. Here we report a regulatory role of Bcl-2 on caspase-2 upstream of mitochondria. Stress stimuli, including ceramide and etoposide, caused caspase-2 activation, mitochondrial damage followed by downstream caspase-9 and -3 activation, and cell apoptosis in human lung epithelial cell line A549. When A549 cells were pretreated with the caspase-2 inhibitor benzyloxycarbonyl-Val-Asp(-OMe)-Val-Ala-Asp(-OMe)-fluoromethyl ketone or transfected with caspase-2 short interfering RNA, both ceramide- and etoposide-induced mitochondrial damage and apoptosis were blocked. Overexpression of Bcl-2 prevented ceramide- and etoposide-induced caspase-2 activation and mitochondrial apoptosis. Furthermore, caspase-2 was activated when A549 cells were introduced with Bcl-2 short interfering RNA or were treated with Bcl-2 inhibitor, which provided direct evidence of a negative regulatory effect of Bcl-2 on caspase-2. Cell survival was observed when caspase-2 was inhibited in Bcl-2-silencing cells. Blockage of the mitochondrial permeability transition pore and caspase-9 demonstrated that Bcl-2-modulated caspase-2 activity occurred upstream of mitochondria. Further studies showed that Bcl-2 was dephosphorylated at serine 70 after ceramide and etoposide treatment. A protein phosphatase inhibitor, okadaic acid, rescued Bcl-2 dephosphorylation and blocked caspase-2 activation, mitochondrial damage, and cell death. Taken together, ceramide and etoposide induced mitochondria-mediated apoptosis by initiating caspase-2 activation, which was, at least in part, regulated by Bcl-2.

Caspase 3, periodically expressed and activated at G2/M transition, is required for nocodazole-induced mitotic checkpoint

Caspases have been known for several years for their involvement in executing apoptosis, where unwanted or damaged cells are eliminated. Surprisingly, after analysis of the relevant data set from the Stanford microarray database, we noticed that the gene expression pattern for caspase 3, but not for caspase 1, 6, 7, 8, 9, or 10, undergoes periodic change in the HeLa cell cycle. In this study, we have demonstrated that caspase 3, but not other caspases, is upregulated and activated just prior to mitosis. Pretreatment of human hepatoma cells with a caspase 3 inhibitor z-DEVD-FMK, prior to the treatment with an antimicrotubule drug nocodazole, abrogates the mitotic arrest, suggesting that caspase 3 (or a caspase 3-like enzyme) might be involved in mitotic-spindle checkpoint. The studies not only characterize caspase 3 as a cell cycle-regulated protein, but also link the protein to nocodazole-dependent mitotic checkpoint, greatly expanding the understanding of caspase 3.

Intracellular alkalinization in dexamethasone-induced thymocyte apoptosis

Glucocorticoid can induce apoptosis of thymocytes, but its mechanism is not clear yet. In this study, we reported that dexamethasone-induced apoptosis was associated with intracellular alkalinization. Dexamethasone induced a higher percentage of apoptosis in 138 mM than in 50 mM NaCl, total abrogation of apoptosis was noted in NaCl-depleted culture medium. Highest apoptotic rate was observed in medium with pH 7.2, whereas it was partially and completely inhibited at pH 6.5 and pH 6.0, respectively. Intracellular pH was higher in pre-apoptotic thymocytes than non-apoptotic ones. The Na+ /H+ antiporter inhibitor of 5-(N,N-dimethyl)-amiloride inhibited the dexamethasone-induced increase in pHi and apoptosis of thymocytes. Glucocorticoid antagonist RU486 also blocked the dexamethasone-induced effect. Furthermore, the apoptosis and increase in intracellular pH induced by dexamethasone were inhibited by cycloheximide, actinomycin D. It seems that intracellular pH is increased during the development of thymocyte apoptosis and inhibiting its increment would retard the rate of progression to cell death.

Activation of caspase‐8 and Erk‐1/2 in domes regulates cell death induced by confluence in MDCK cells

Under normal culture conditions, cells adhere to culture dish, spread out, proliferate, and finally cover all areas and reach confluence. During the confluent stage, cell proliferation ceases and differentiation is enhanced. Meanwhile, cell death also appears as the monolayer confluence proceeds. To delineate the mechanism of cell death induced by the confluent process, we employed Madin‐Darby canine kidney (MDCK) cells. When approaching confluence, MDCK cells exhibited increase the levels of caspase‐2 and enhanced the activity of caspase‐8. Using various caspase inhibitors to block apoptosis, we found that only z‐VAD‐fmk and z‐IETD‐fmk can inhibit confluent cell death, indicating that confluent cell death is mediated by activation of caspase‐8. Overexpression of Bcl‐2 inhibited confluent cell death, suggesting the involvement of mitochondria‐dependent pathway in confluent cell death. Interestingly, the activity of phospho‐Erk (p‐Erk) was initially decreased before confluence, but markedly increased after confluence. Immunofluorescence staining studies showed that p‐Erk was expressed exclusively on dome‐forming cells that underwent apoptosis. Treatment of confluent MDCK cells with PD98059 and UO126, the inhibitors of MEK, enhanced apoptosis as well as activity of caspase‐8. These data indicate that elevation of p‐Erk activity during confluence may serve to suppress confluent cell death. Taken together, activation of caspase‐8 contributes to and results in confluent cell death, whereas elevated p‐Erk activity serves to prevent confluent cell death by regulating activation of caspase‐8. J. Cell. Physiol. 211: 174–182, 2007.