Non-Selective Inhibition of Transformed Cell Growth by a Protease Inhibitor

Chou, Iih‐Nan; Black, Paul H.; Roblin, Richard O.

doi:10.1073/pnas.71.5.1748

Cited by 36 publications

(10 citation statements)

References 20 publications

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“…It has been suggested that protease inhibitors suppress cell growth by inhibiting protein synthesis directly (31). However, Grayzel et al (20) demonstrated that TLCK significantly inhibits lymphocyte transformation without a major effect on protein synthesis.…”

Section: Methodsmentioning

confidence: 99%

Induction of DNA synthesis in isolated nuclei by cytoplasmic factors: inhibition by protease inhibitors.

Wong¹,

Gutowski²,

Katz³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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Cytoplasmic extracts from spontaneously proliferating and mitogen-activated lymphoid cells contain a protein factor called ADR (activator of DNA replication) that induces DNA synthesis in isolated quiescent nuclei. ADRcontaining preparations have proteolytic activity, as indicated by their ability to degrade fibrin in a plasminogen-independent and plasminogen-dependent manner. In addition, aprotinin, a nonspecific protease inhibitor, abrogates ADR-induced DNA synthesis in a dose-dependent fashion. Preincubation studies demonstrated that the effect of aprotinin is not due to its suppressive effects on the nuclei themselves. Other protease inhibitors such as leupeptin, p-aminobenzamidine, and N-atosyllysine chloromethyl ketone are also inhibitory, but soybean trypsin inhibitor is without effect. ADR activity can be removed from active extracts by adsorption with aprotininconjugated agarose beads and can be recovered by elution with an acetate buffer (pH 5). These findings are consistent with the interpretation that the initiation of DNA synthesis in resting nuclei may be protease dependent and, further, that the cytoplasmic stimulatory factor we have called ADR may be a protease itself.It has been known for many years that nuclear DNA synthesis is under cytoplasmic control (1). Using cell-free systems, a number of investigators have shown that cytoplasmic extracts from proliferating cells contain a protein(s) that can initiate nuclear DNA synthesis (2-9). We have reported such cytoplasmic intermediate(s) in spontaneously proliferating and mitogen-activated lymphoid cells (7,8). This factor, which we have called activator of DNA replication (ADR), is a heat-labile protein of Mr >100,000. It is not detectable in resting cells. Although it activates isolated quiescent nuclei, it has no effect on intact cells. ADR is not species specific. Furthermore, ADR appears to mediate an intracellular mitogenic signal in the interleukin 2 (IL-2) T-cell activation pathway (9). Das (6) has described a cytoplasmic intermediate in epidermal growth factor-stimulated 3T3 fibroblasts that shares many functional and physicochemical properties with ADR. In addition, Benbow and Ford (2) reported a similar cytoplasmic stimulatory factor in early embryonic tissue. The description of ADR-like proteins in a number of cell types and experimental systems suggests a significant role for these factors in cellular proliferation and differentiation.To our knowledge, no information is available as to the biochemical nature of ADR or the ADR-like factors in other cell types (2-6). We now report that ADR preparations have proteolytic activity and that the functional activity of ADR can be significantly inhibited by aprotinin and a number of other protease inhibitors. The inhibition of ADR-induced DNA synthesis was not due to any direct effects of aprotinin on the isolated resting nuclei. Rather, the protease inhibitor appears to interact with ADR itself. MATERIALS AND METHODSCell Cultures and Preparation of Cytoplasmic Extracts. MOLT-4, a sp...

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Section: Methodsmentioning

confidence: 99%

Induction of DNA synthesis in isolated nuclei by cytoplasmic factors: inhibition by protease inhibitors.

Wong¹,

Gutowski²,

Katz³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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“…Earlier workers studied the effects of protease inhibitors such as 5-amino-L-1-tosylamidopentyl chloromethyl ketone ("tosyllysyl chloromethyl ketone," TLCK) and L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) on growth of normal and transformed cells, in particular the suppression of contact inhibition (1)(2)(3). However, it was found that the growth-inhibiting effect was due, in large part, to the suppression of macromolecular synthesis (4). A highly specific inhibitor with fewer side effects is required for detailed investigation of the function of proteases in eukaryotic cells.…”

mentioning

confidence: 99%

Thiol protease-specific inhibitor E-64 arrests human epidermoid carcinoma A431 cells at mitotic metaphase.

Shoji-Kasai

Senshu

Iwashita

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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E-64-d {ethyl (2S, 3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)butylcarbamoylloxirane-2-carboxylate}, a membrane-permeant derivative of the thiol proteasespecific inhibitor E-64, was found to arrest human epidermoid carcinoma A431 cells at mitotic metaphase. This effect was dose-dependent with a threshold of 20 ,ug/ml in chemically defined culture medium. Cell cycle analysis by flow cytometry showed that the relative proportion of the G2/M population increased 2.5-fold after treatment of the cells with E-64 (100 ,g/ml) for 5 hr. In addition, time-lapse video analysis showed that E-64-treated cells remained at metaphase for an extended period after rounding-up, whereas untreated cells completed mitosis within 42.0 ± 5.7 min. Some treated cells were able to complete mitosis, while others did not do so within the limits of our observation. As an approach to the molecular basis of this phenomenon, we have shown that several cellular proteins can be labeled by incubation of cells with radioactive E-64-d.

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“…As S6K1 mediates important events for cell growth and proliferation (32)(33)(34), the immunosuppressant activity of rapamycin may be due, at least in part, to its inhibition of S6K1. To more fully understand the biological role of S6K1, we previously evaluated the effect of the enzymatic inhibitors N-␣-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) and N-␣-tosyl-L-lysyl chloromethyl ketone (TLCK) on S6K1 activation given their known anti-proliferative and anti-tumorigenic capacity (35)(36)(37)(38)(39)(40). Interestingly, like rapamycin, TPCK (and less potently, TLCK) inhibited the activation of S6K1 by multiple agonists.…”

mentioning

confidence: 99%

Disruption of 3-Phosphoinositide-dependent Kinase 1 (PDK1) Signaling by the Anti-tumorigenic and Anti-proliferative AgentN-α-tosyl-l-phenylalanyl Chloromethyl Ketone

Ballif

Shimamura

Pae

et al. 2001

Journal of Biological Chemistry

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The anti-tumorigenic and anti-proliferative effects of N-␣-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) have been known for more than three decades. Yet little is known about the discrete cellular targets of TPCK controlling these effects. Previous work from our laboratory showed TPCK, like the immunosuppressant rapamycin, to be a potent inhibitor of the 70-kilodalton ribosomal S6 kinase 1 (S6K1), which mediates events involved in cell growth and proliferation. We show here that rapamycin and TPCK display distinct inhibitory mechanisms on S6K1 as a rapamycin-resistant form of S6K1 was TPCK-sensitive. Additionally, we show that TPCK inhibited the activation of the related kinase and proto-oncogene Akt. Upstream regulators of S6K1 and Akt include phosphoinositide 3-kinase (PI 3-K) and 3-phosphoinositide-dependent kinase 1 (PDK1). Whereas TPCK had no effect on either mitogen-regulated PI 3-K activity or total cellular PDK1 activity, TPCK prevented phosphorylation of the PDK1 regulatory sites in S6K1 and Akt. Furthermore, whereas both PDK1 and the mitogen-activated protein kinase (MAPK) are required for full activation of the 90-kilodalton ribosomal S6 kinase (RSK), TPCK inhibited RSK activation without inhibiting MAPK activation. Consistent with the capacity of RSK and Akt to mediate a cell survival signal, in part through phosphorylation of the pro-apoptotic protein BAD, TPCK reduced BAD phosphorylation and led to cell death in interleukin-3-dependent 32D cells. Finally, in agreement with results seen in embryonic stem cells lacking PDK1, protein kinase A activation was not inhibited by TPCK showing TPCK specificity for mitogen-regulated PDK1 signaling. TPCK inhibition of PDK1 signaling thus disables central kinase cascades governing diverse cellular processes including proliferation and survival and provides an explanation for its striking biological effects.

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Non-Selective Inhibition of Transformed Cell Growth by a Protease Inhibitor

Cited by 36 publications

References 20 publications

Induction of DNA synthesis in isolated nuclei by cytoplasmic factors: inhibition by protease inhibitors.

Induction of DNA synthesis in isolated nuclei by cytoplasmic factors: inhibition by protease inhibitors.

Thiol protease-specific inhibitor E-64 arrests human epidermoid carcinoma A431 cells at mitotic metaphase.

Disruption of 3-Phosphoinositide-dependent Kinase 1 (PDK1) Signaling by the Anti-tumorigenic and Anti-proliferative AgentN-α-tosyl-l-phenylalanyl Chloromethyl Ketone

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