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Zbigniew Darzynkiewicz

Publications and source records attributed to Zbigniew Darzynkiewicz.

86 records · Page 5Linked to original sources
PubMed2002

Histone deacetylase inhibitors all induce p21 but differentially cause tubulin acetylation, mitotic arrest, and cytotoxicity.

By preventing deacetylation of histones, histone deacetylase inhibitors (HDIs) transcriptionally induce p21. Here we show that the HDIs sodium butyrate (Bu), trichostatin A (TSA) and depsipeptide (FR901228) all induced p21, but only TSA and FR901228 caused mitotic arrest (in addition to arrest in G1 and G2). The ability to cause mitotic arrest correlated with the higher cytotoxicity of these compounds. Although causing mitotic arrest, TSA and FR901228 (unlike paclitaxel) did not affect tubulin polymerization. Unlike FR9012208, TSA caused acetylation of tubulin at lysine 40; both soluble tubulin and microtubules were acetylated. Whereas the induction of p21 reached a maximum by 8 h, tubulin was maximally acetylated after only 1 h of TSA treatment. Tubulin acetylation was detectable after treatment with 12-25 ng/ml TSA although acetylation plateaued at 50 ng/ml TSA, coinciding with G2-M arrest, appearance of cells with a sub-2N DNA content, poly(ADP-ribose) polymerase cleavage, and rapid cell death. We conclude that HDIs have differential effects on non-histone deacetylases and that rapid acetylation of tubulin caused by TSA is a marker of nontranscriptional effects of TSA.

Anesthetics, Intravenous↗
PubMed2001

ASB-2 inhibits growth and promotes commitment in myeloid leukemia cells.

In acute promyelocytic leukemia (APL) cells harboring the promyelocytic leukemia retinoic acid receptor alpha (PML-RARalpha) chimeric protein, retinoic acid (RA)-induced differentiation is triggered through a PML-RARalpha signaling resulting in activation of critical target genes. Induced differentiation of APL cells is always preceded by withdrawal from the cell cycle and commitment events leading to terminal differentiation. Here we have identified the human ankyrin repeat-containing protein with a suppressor of cytokine signaling box-2 (ASB-2) cDNA, as a novel RA-induced gene in APL cells. PML-RARalpha strongly enhanced RA-induced ASB-2 mRNA expression. In myeloid leukemia cells, ASB-2 expression induced growth inhibition and chromatin condensation recapitulating early events critical to RA-induced differentiation of APL cells.

Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

Flavopiridol inversely affects p21(WAF1/CIP1) and p53 and protects p21-sensitive cells from paclitaxel.

Resting cells are relatively resistant to microtubule-active drugs including paclitaxel (PTX). By causing p53-mediated arrest, pretreatment with low concentrations of doxorubicin (DOX) protected HCT116 cells from the cytotoxicity caused by PTX. Unlike DOX, flavopiridol (FL) did not protect HCT116 cells. Low concentrations of FL (50 nM) induced p21 but not p53. High concentrations of FL (500 nM) decreased levels of p21 and Mdm-2 but dramatically induced p53. Thus, FL reciprocally affects p21 and p53. In LNCaP, a prostate cancer cell line which is highly sensitive to p21-induced growth arrest (p21-sensitive), low concentrations of FL (50 nM) induced p21 (without induction of p53) and caused G1 and G2 arrest. This precluded mitotic arrest, Bcl-2 and Raf-1 phosphorylation, and diminished cell death caused by PTX. In contrast, FL did not protect PC3M, arrest-resitant and highly aggressive prostate cancer cells. Like LNCaP, HL60 and SKBr3 cells are known to be p21-sensitive. As predicted, low concentrations of FL antagonized PTX-mediated cytotoxicity in HL60 and SKBr3 cell lines. In summary, only low concentrations of FL can induce p21, and, in turn, only p21-sensitive cells are protected from PTX.

Antineoplastic Agents↗
PubMedDate not supplied

Why Iressa failed: toward novel use of kinase inhibitors (outlook).

A phase III failure of Iressa, an inhibitor of the EGF receptor (EGFR), is viewed as a surprise. With a few exceptions, however, inhibitors of EGFR cannot be effective as a monotherapy in cancers with additional oncogenic changes (downstream of EGFR), which cause mitogen-independent proliferation. In other cases, combining these inhibitors with chemotherapy may lead to antagonism in cancer cells and/or aggravated side-effects. This review discusses why neither levels of EGFR nor doses of Iressa correlate with clinical response. We suggest a novel use of signal transduction inhibitors, including Iressa, to increase therapeutic index by modulating cycle-dependent and apoptosis-inducing chemotherapies. This approach may be most beneficial to patients who do not respond to monotherapy with kinase inhibitors. Development of molecular diagnostics will further diversify these strategies.

Antineoplastic Agents↗
PubMedDate not supplied

Cyclotherapy: protection of normal cells and unshielding of cancer cells.

Avoidance of apoptosis and mitogen-independent growth are hallmarks of cancer. Mitogen-activated kinases (for example, ErbB1, Raf-1, MEK, PI-3-K, mTOR) can suppress chemotherapy-induced apoptosis in cancer cells. While kinase inhibitors restore susceptibility of cancer cells to apoptosis, they do not necessarily cause growth arrest in cancer cells harboring additional mutations in downstream signaling pathways such as inactivation of Rb and overexpression of c-myc. This article provides a conceptual basis for a novel use of inhibitors of mitogenic kinases. While arresting growth of normal cells, kinase inhibitors may not arrest cancer cells but instead can sensitize them to apoptosis. Following pretreatment with low doses of kinase inhibitors, the chemotherapy that predominantly induces apoptosis in cycling cells (cyclotherapy) will kill cancer cells while sparing normal cells.

Animals↗
PubMedDate not supplied

Cytotoxic ribonucleases and RNA interference (RNAi).

Several cytotoxic ribonucleases (CRs), homologs of the pancreatic RNase A, have been isolated from amphibian oocytes or embryos. Of them, onconase (Onc), the CR that shows antitumor properties and is in phase III clinical trials, was the most extensively researched. Degradation of tRNA by Onc internalized into cells that leads to inhibition of protein synthesis is considered the mechanism of its cytotoxicity. Several findings, however, cannot be explained by nonspecific decline in protein synthesis alone and suggest additional or alternative mechanism(s). We postulate therefore that miRNAs and/or RNA interference (RNAi) may also be targets of CRs. The following arguments support this postulate: (A) miRNAs and siRNAs appear to be unprotected by proteins and therefore, as tRNA, accessible and degradable by CRs; (B) Onc has preferred cleavage sites on tRNAs: their cleavage may generate segments of dsRNA that interfere with translation. Analogous to Dicer, thus, small RNAs with interfering properties may be generated by CRs within the cell; (C) CRs are abundant in oocytes and during embryonic development; their role there is unknown. Since cells undergo perpetual differentiation during embryogenesis it is likely that the function of CRs is to provide additional level of regulation of gene expression via the mechanisms listed in (A) and/or (B).

Animals↗
PubMedDate not supplied

Effects of PC-SPES on proliferation and expression of AR/PSA in androgen-responsive LNCaP cells are independent of estradiol.

Previous studies have suggested that the clinical efficacy of PC-SPES, a dietary supplement used frequently by men diagnosed with androgen-dependent (AD) or androgen-independent (AI) prostate cancer (CaP), is mechanistically attributed to estrogenic components present in the herbal mixture. To test this hypothesis, we compared estradiol (1 nM), potentially an active principle in PC-SPES, with PC-SPES (using an amount equivalent to 1 nM estradiol) on cell proliferation, induction of apoptosis, and regulation of prostate specific genes, PSA and AR, in androgen-responsive LNCaP cells. Cells cultured in steroid-proficient (FBS) or-deficient (CS-FBS) media to simulate hormonal status pre- and post-castration in vivo, were incubated with estradiol or PC-SPES. Proliferation was reduced in PC-SPES treated cells cultured in media supplemented with FBS or CS-FBS; in contrast, addition of estradiol had no effect on proliferation in FBS cultures, and elicited a 45% growth increase in CS-PBS-supplemented cultures. The differential proliferative response of LNCaP cells to PC-SPES vs. estradiol was also supported by changes in PCNA expression, cell viability, cell cycle phase distribution, and induction of apoptosis. Estradiol elicited time-dependent increases in secreted PSA, whereas PC-SPES suppressed PSA secretion, in both culture conditions. In FBS cultures, PC-SPES lowered intracellular AR and PSA by 61% and 17%, respectively, while estradiol increased intracellular PSA, in parallel with a 42% decrease in AR expression. In comparison with cells maintained with CS-FBS, estradiol induced substantial increases in both intracellular PSA and AR, whereas PC-SPES resulted in a smaller increase in intracellular PSA without affecting the expression of AR. These studies show that the antiproliferative and gene modulatory effects of PC-SPES in androgen-dependent human prostate cancer cells are mechanistically and functionally distinct from effects attributable to estradiol.

Antineoplastic Agents, Phytogenic↗
PubMedDate not supplied

Sequential activation of caspases and serine proteases (serpases) during apoptosis.

Analogous to caspases, serine (Ser) proteases are involved in protein degradation during apoptosis. It is unknown, however, whether Ser proteases are activated concurrently, sequentially, or as an alternative to the activation of caspases. Using fluorescent inhibitors of caspases (FLICA) and Ser proteases (FLISP), novel methods to detect activation of these enzymes in apoptotic cells, we demonstrate that two types of Ser protease sites become accessible to these inhibitors during apoptosis of HL-60 cells. The prior exposure to caspases inhibitor Z-VAD-FMK markedly diminished activation of both Ser protease sites. However, the unlabeled inhibitor of Ser-proteases TPCK had modest suppressive effect- while TICK had no effect- on the activation of caspases. Activation of caspases, thus, appears to be an upstream event and likely a prerequisite for activation of FLISP-reactive sites. Differential labeling with the red fluorescing sulforhodamine-tagged VAD-FMK and the green fluorescing FLISP allowed us to discriminate, within the same cell, between activation of caspases and Ser protease sites. Despite a certain degree of co-localization, the pattern of intracellular caspase- vs FLISP- reactive sites, was different. Also different were relative proportions of activated caspases vs Ser protease sites in individual cells. The observed induction of FLISP-binding sites we interpret as revealing activation of at least two different apoptotic Ser proteases; by analogy to caspases we denote them serpases. Their apparent molecular weight (62-65 kD) suggests that they are novel enzymes.

Amino Acid Chloromethyl Ketones↗
PubMedDate not supplied

Arsenic trioxide arrests cells early in mitosis leading to apoptosis.

Arsenic trioxide (As(2)O(3)) is highly effective in the treatment of acute promyelocytic leukemias that express the promyelocytic leukemia-retinoic acid receptor-alpha (PML-RARalpha) fusion protein. However, evidence has accumulated that As(2)O(3) induces apoptosis regardless of PML-RARalpha status. Here we show that, at clinically relevant concentrations, As(2)O(3) causes S and G(2)M phase arrest of both PML-RARalpha-positive and -negative leukemia cell lines, thus inhibiting their growth. Apoptotic cells are generated predominately from the G(2)M fraction. Using several independent methods, we demonstrate that the cells accumulated in the G(2)M peak consist primarily of cells arrested in the early stages of mitosis, prophase, prometaphase and metaphase. In mitotic cells, there was significant activation of caspases, PARP cleavage, and morphological changes characteristic of apoptosis. Unlike microtubule-active drugs that arrest cells in metaphase, arsenic trioxide did not affect the architecture of microtubules. Our data suggest that the antileukemic activities of arsenic may be a result of mitotic arrest which culminates in apoptosis.

Antineoplastic Agents↗
PubMedDate not supplied

Cell cycle effects and caspase-dependent and independent death of HL-60 and Jurkat cells treated with the inhibitor of NF-kappaB parthenolide.

The sesquiterpene parthenolide (PRT) is an active component of Mexican-Indian medicinal plants and also of the common herb of European origin feverfew. PRT is considered to be a specific inhibitor of NF-kappaB. Human leukemic HL-60, Jurkat, and Jurkat IkappaBalphaM cells, the latter expressing a dominant-negative IkappaBalpha and thus having non-functional NF-kappaB, were treated with PRT and activation of caspases, plasma membrane integrity, DNA fragmentation, chromatin condensation (probed by DNA susceptibility to denaturation), and changes in cell morphology were determined. As a positive control for apoptosis cells were treated with topotecan (TPT) and H2O2. At 2-8 microM concentration PRT induced transient cell arrest in G2 and M followed by apoptosis. A narrow range of PRT concentration (2-10 microM) spanned its cytostatic effect, induction of apoptosis and induction of necrosis. In fact, necrotic cells were often seen concurrently with apoptotic cells at the same PRT concentration. Atypical apoptosis was characterized by loss of plasma membrane integrity very shortly after caspases activation. In contrast, a prolonged phase of caspase activation with preserved integrity of plasma membrane was seen during apoptosis induced by TPT or H2O2. Necrosis induced by PRT was also atypical, characterized by rapid rupture of plasma membrane and no increase in DNA susceptibility to denaturation. Using Jurkat cells with inactive NF-kappaB we demonstrate that cell cycle arrest and the mode of cell death induced by PRT were not caused by inhibition of NF-kappaB. The data suggest that regardless of caspase activation PRT targets plasma membrane causing its destruction. A caution, therefore, should be exercised in interpreting data of the experiments in which PRT is used with the intention to specifically prevent activation of NF-kappaB.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMedDate not supplied

DNA damage induced by DNA topoisomerase I- and topoisomerase II-inhibitors detected by histone H2AX phosphorylation in relation to the cell cycle phase and apoptosis.

Histone H2AX is phosphorylated on Ser-139 by ATM kinase in response to damage that induces dsDNA breaks. Immunocytochemical detection of phosphorylated H2AX (gammaH2AX), thus, reveals the presence of dsDNA breaks in chromatin. Multiparameter cytometry was presently used to correlate the appearance of gammaH2AX with: a. cell cycle phase; b. caspase-3 activation; and c. apoptosis-associated DNA fragmentation in individual human leukemic HL-60 cells treated with the DNA topoisomerase I (topo1) inhibitors topotecan (TPT) and camptothecin (CPT) or with the topo2 inhibitor mitoxantrone (MTX). In response to TPT or CPT maximal increase of gammaH2AX immunofluorescence was seen in S-phase cells by 90 min. In contrast, following MTX treatment the maximal rise of gammaH2AX was detected at 2 h in G1 cells and the cell cycle phase specificity was much less apparent. A linear relationship between the drug concentration and increase of gammaH2AX immunofluorescence was seen only up to 200 nM TPT; a decline in gammaH2AX was apparent at a concentration range between 0.4 and 1.6 microM TPT. Thus, the intensity of gammaH2AX immunofluorescence, as a marker of cell survival following TPT treatment, can be used only within a limited range of drug concentration. Following treatment with TPT, CPT or MTX the peak of H2AX phosphorylation preceded caspase-3 activation and the appearance of apoptosis-associated DNA fragmentation, both selective to S-phase cells. Progression of apoptosis was paralleled by a decrease in gammaH2AX immunofluorescence. The data also indicate that regardless whether treated with inhibitors of topo1 or topo2, at comparable levels of dsDNA breaks, the cells replicating DNA have a higher proclivity to undergo apoptosis compared to G1 or G2/M cells.

Apoptosis↗
PubMedDate not supplied

High frequency of allele-specific down-regulation of HLA class I expression in lung cancer cell lines.

Loss or down-regulation of HLA expression has been demonstrated in various types of solid tumors and is considered to be one of the mechanisms of tumor immunoescape. The effectiveness of immunotherapy using tumor-specific antigens (TSA) largely depends on the expression of the appropriate HLA class I alleles on the tumor cells. We analyzed the allele-specific HLA class I surface expression of six lung cancer cell lines using a broad panel of allele-specific monoclonal antibodies, as well as the effect of IFN-gamma on HLA expression. Flow cytometric analysis displayed a wide range, from minimal to a high degree of expression of monomorphic HLA class I among the studied cell lines. Allele-specific loss or down-regulation of HLA also was observed in 5 out of 6 cell lines. Our results suggest that lung cancer patients considered for specific immunotherapy should be examined with respect to the expression of specific HLA class I allele binding the TSA.

Alleles↗