P21 Waf1 Research Articles

Enhancement of chemotherapeutic drug-induced apoptosis by a cyclooxygenase-2 inhibitor in hypopharyngeal carcinoma cells.

Our previous study demonstrated that cyclooxygenase-2 (COX-2) was overexpressed in human hypopharyngeal carcinoma. In this study, we tested the effect of a specific COX-2 inhibitor, NS398, on proliferation of hypopharyngeal cancer cells. Our results indicated that NS398 inhibited growth of hypopharyngeal cancer cells and this inhibition is associated with induction of G1 growth arrest. Western blot analysis showed that expression of G1 cyclins or cyclin-dependent kinases (CDKs) was not changed by NS398. On the contrary, NS398 significantly increased expression of CDK inhibitors p21(Waf1) and p27(Kip1). We also found that treatment of NS398 alone could not induce significant apoptosis in hypopharyngeal cancer cells. However, NS398 potently augmented chemotherapeutic drug-induced apoptosis. Caspase activation and DNA fragmentation were clearly detected in cancer cells pretreated with NS398 followed by chemotherapeutic drugs. Collectively, our results suggest that COX-2 inhibitors may suppress proliferation of hypopharyngeal cancer cells via induction of G1 growth arrest and may be useful in combination with chemotherapeutic drugs for the treatment of hypopharyngeal carcinoma.

Inhibition of JAK3 induces apoptosis and decreases anaplastic lymphoma kinase activity in anaplastic large cell lymphoma.

Signal transducer and activator of transcription 3 (STAT3), normally activated by Janus kinase (JAK) in response to cytokine stimulation, has been shown to have oncogenic potential. In addition to JAK, recent data suggest that STAT3 can also be activated by other proteins such as the aberrant fusion protein, NPM-ALK, which is expressed in a subset of systemic anaplastic large cell lymphoma (ALCL). In this study, we investigated the possible role of JAK in activating STAT3 in ALCL using two ALK-positive ALCL cell lines, Karpas 299 and SU-DHL-1. At the steady state, JAK3 showed detectable tyrosine phosphorylation by immunoprecipitation. Treatment with AG490, a JAK inhibitor, decreased but did not completely abrogate tyrosine phosphorylation of JAK3 and STAT3 in a concentration-dependent manner. Similar results were obtained using two other inhibitors of JAK3, WHI-P131 and WHI-P154. These biochemical changes were associated with apoptosis in both cell lines that was coupled with activation of caspase 3 and decreased bcl-xL and bcl-2. Cell cycle analysis revealed a decrease in the S phase, which may be attributed to cyclin D3 downregulation and p21(waf1) upregulation. Importantly, the tyrosine kinase activity of NPM-ALK, as assessed by an in vitro assay, decreased with increasing concentrations of AG490. Our findings highlight the importance of JAK3 in activating STAT3 in ALCL, and that NPM-ALK-mediated activation of STAT3 is influenced by the functional status of JAK3.

Tissue-specific p53 responses to ionizing radiation and their genetic modification: the key to tissue-specific tumour susceptibility?

Although little is understood of the underlying mechanisms, there are tissue-specific responses to tumourigenic and therapeutic agents and these responses are influenced by genetic factors. Ionizing radiation is an important tumourigenic and therapeutic agent for which there is substantial evidence for such tissue-dependent and genotype-dependent responses. Because the p53 tumour suppressor protein is a major determinant of cellular responses to radiation, the present study has investigated whether modification of the p53 pathway contributes to tissue-dependent and genotype-dependent responses using inbred strains of mice. Comparison of responses in haemopoietic and epithelial cells in irradiated C57BL/6 and DBA/2 mice revealed significant differences in p53 and apoptotic responses in different cell types and in different cells of the same type, reflecting the complexity of damage responses operating in the whole organism. The data suggest that p53-mediated up-regulation of Bax is a major determinant of apoptosis in the spleen, but not in the intestine, whereas p53-mediated induction of p21(waf1) plays an anti-apoptotic role in the spleen, but not in the intestine. It is also shown that p53 stabilization and differential transactivational activities towards Bax or p21(waf1) are influenced by genetic factors that act in a tissue-specific manner. Analysis of ATM, a potential mediator of differential p53 activation, indicates that this key regulator of radiation responses is preferentially induced in epithelial cells, but is unlikely to account for genetic modification of p53 or apoptotic responses in the mouse strains studied. Polymorphisms in the p53 or DNA-PKcs genes are also unlikely to account for the genetic modifications that are reported here. There are numerous further potential modifiers of the p53 pathway, but analysis of backcross and inter-cross mice demonstrates that genes responsible for the complex modification of these in vivo responses can be identified by linkage analysis. This approach has the potential to reveal new or unexpected interactions involving the p53 pathway that determine both short-term and long-term effects of radiation exposure and the basis of tissue-specific responses and tumour susceptibility.

Down-Regulation of Proliferating Cell Nuclear Antigen Gene Expression Occurs during Cell Cycle Arrest Induced by Human Fecal Water in Colonic HT-29 Cells

Cancer is a disease in which the cell cycle is altered, and the elucidation of the mechanisms by which constituents of human fecal water influence the cell cycle can lead to noninvasive measurement of colon cancer risk. The purpose of the present study was to investigate the effect of human fecal water on HT-29 cell-cycle progression with sodium selenite as a reference for comparison. Both human fecal water (2.5-5.0%) and selenite (3-4 micro mol/L) significantly inhibited cell growth. Cell-cycle analysis revealed that human fecal water decreased the proportion of S + G2 phase cells and increased that of G1 phase cells. In contrast, selenite decreased G1 phase cells and increased proportions of S and G2 phase cells. Both 5% human fecal water and 4 micro mol/L selenite greatly increased the mRNA level of the cyclin-dependent kinase inhibitor gene p21(waf1). Interestingly, the mRNA levels of cyclin A and proliferating cell nuclear antigen (PCNA) were dramatically decreased by 69 and 62%, respectively, in HT-29 cells treated with fecal water but not selenite. In contrast, the mRNA level of DNA damage-inducible transcript 1, gadd45, was significantly increased by 2.28-fold in HT-29 cells treated with selenite but not fecal water. Furthermore, a PCNA gene promoter was cloned into a luciferase reporter construct and its activity was significantly reduced in a dose-dependent manner in cells treated with fecal water but not selenite. Collectively, these results suggest that human fecal water and selenite can differentially induce growth arrest genes, and that PCNA gene expression is uniquely and highly sensitive to human fecal water.

ZBP-89 Mediates Butyrate Regulation of Gene Expression

Inducible p53-independent regulation of the cyclin-dependent kinase inhibitor p21(Waf1) transcription is mediated through its proximal GC-rich sites. Prior studies have shown that Sp1, Sp3 and the histone acetyltransferase coactivator p300 are components of the complexes that bind to these sites. Although Sp1 and Sp3 collaborate with p300, a direct interaction between Sp1 and p300 does not occur. Zinc-finger binding protein-89 (ZBP-89, also known as BFCOL1, BERF-1 and ZNF-148) is a Krüppel-type zinc-finger transcription factor that binds to the same GC-rich sequences as Sp1. We sought to determine whether ZBP-89 is a target of p300 during butyrate induction of p21(Waf1). This review summarizes the evidence that supports a crucial role for ZBP-89 in butyrate regulation of p21(Waf1). Adenovirus-mediated expression of ZBP-89 in HT-29 cells reveals that ZBP-89 potentiates butyrate induction of endogenous p21(Waf1) gene expression. DNA-protein interaction assays demonstrate that Sp1, Sp3 and ZBP-89 bind the p21(Waf1) promoter at -245 to -215. Coprecipitation assays reveal that p300 preferentially binds to the N-terminus of ZBP-89. ZBP-89 also induces p21(Waf1) through stabilization of p53. Although ZBP-89 binds mutant and wild-type p53, only wild-type p53 is stabilized. Moreover, mutant p53 shifts the subnuclear location of ZBP-89 to the nuclear periphery, which is a domain rich in heterochromatin. This finding led to the conclusion that mutant p53 exerts a dominant negative effect on ZBP-89. We propose that gene silencing by mutant p53 might be mediated by sequestering ZBP-89 within heterochromatin regions at the nuclear periphery. Overall, ZBP-89 is a butyrate-regulated coactivator of p53 and is able to induce p21(Waf1) gene expression through both p53-dependent and -independent mechanisms to inhibit cell growth.

Accumulation of p27 KIP1 is associated with BMP2-induced growth arrest and neuronal differentiation of human neuroblastoma-derived cell lines

Bone morphogenetic proteins (BMPs) play an essential role in cell fate determination. In this study, we found that BMP2 treatment resulted in growth arrest and differentiation in human neuroblastoma-derived cell lines, SH-SY5Y and RTBM1. Within 30 min of BMP2 exposure, phosphorylation of Smad1/5 was observed in these cell lines. In RTBM1 cells, BMP2-induced differentiation was accompanied by a significant decrease in the expression level of DAN, an antagonist of BMP in frog embryos. Immunoblot analysis revealed that BMP2 treatment caused a down-regulation of p53 family members and hence of cyclin-dependent kinase inhibitor p21 WAF1. We found a significant accumulation of p27 KIP1 in response to BMP2, whereas the expression level of Skp2, which is required for ubiquitin-dependent p27 KIP1 degradation, was decreased during this differentiation process. Our results suggest that p27 KIP1 contributes to the BMP-induced growth arrest and neuronal differentiation of neuroblastoma, and BMP treatment might provide a new therapeutic strategy.

Sensitization of human breast cancer cells to gemcitabine by the protein kinase C modulator bryostatin 1.

Protein kinase C (PKC) plays an important role in cell proliferation, differentiation, and apoptosis. The interaction between the PKC modulator bryostatin 1 (BRYO), and gemcitabine in human breast cancer MCF-7 and MDA-MB-231 cells and in the non-transformed MCF-10A human breast epithelial cells was investigated. Immunoblotting was used to determine the expression of PKC isoenzymes and proteins involved in the cell cycle and apoptosis. MTT, ELISA and flow cytometry assays were used to determine cell survival. Treatment of cells with BRYO 200 n M resulted in a significant downregulation of cytoplasmic PKC in all three cell lines. However, the expression of membranous PKC was differentially affected in these cells. BRYO (1-200 n M) had no significant effects on cell viability in any of the cell lines. Nevertheless, BRYO significantly enhanced the antiproliferative and apoptotic effects of gemcitabine in the MCF-7 and MDA-MB-231 cells, but not in the MCF-10A cells. This was associated with significant reduction in the bcl-2/bax ratio. There was a significant upregulation of p53, p21(waf1), and p27 in MCF7 and MCF-10A cells treated with the combination of gemcitabine and BRYO compared to gemcitabine-treated cells. The potentiation of the effect of gemcitabine by BRYO was demonstrated in MCF-7 and MDA-MB-231 cells and was associated with a specific pattern of PKC modulation. Further investigation of the role of specific isoforms of PKC in the downstream molecular events of gemcitabine-induced cytotoxicity is warranted.

Repression of TGF-beta signaling by the oncogenic protein SKI in human melanomas: consequences for proliferation, survival, and metastasis.

Transforming growth factor-beta (TGF-beta ) has dual and paradoxical functions as a tumor suppressor and promoter of tumor progression and metastasis. TGF-Ji-mediated growth inhibition is gradually lost during melanoma tumor progression, but there are no measurable defects at the receptor level. Furthermore, melanoma cells release high levels of TGF-beta to the microenvironment, which upon activation induces matrix deposition, angiogenesis, survival, and transition to more aggressive phenotypes. The SKI and SnoN protein family associate with and repress the activity of Smad2, Smad3, and Smad4, three members of the TGF-fl signaling pathway. SKI also facilitates cell-cycle progression by targeting the RB pathway by at least two ways: it directly associates with RB and represses its activity when expressed at high levels, and indirectly, it represses Smad-mediated induction of p21(Waf-1) This results in increased CDK2 activity, RB phosphorylation,and inactivation. Therefore, high levels of SKI result in lesions to the RB pathway in a manner similar to p16 (INK4a) loss. SKI mRNA and protein levels dramatically increase during human melanoma tumor progression. In addition,the SKI protein shifts from nuclear localization in intraepidermal melanoma cells to nuclear and cytoplasmic in invasive and metastatic melanomas. Here, I discuss the basis for repression of intracellular TGF-beta signaling by SKI, some additional activities of this protein, and propose that by disrupting multiple tumor suppressor pathways, SKI functions as a melanoma oncogene.

Decitabine induces cell cycle arrest at the G1 phase via p21 WAF1 and the G2/M phase via the p38 MAP kinase pathway

Methylation of the p16 INK4a tumor suppressor gene is observed frequently in multiple myeloma and various forms of lymphoma and mediates silencing of p16 gene expression. In this investigation, we have determined the effect of the DNA demethylating drug decitabine (DAC; 5-aza-2′-deoxycytidine) on the growth, cell cycle kinetics, RB phosphorylation, and expression of p16 INK4a and p21 WAF1 in EBV− human myeloma and EBV+ lymphoblastic cell lines possessing silenced, methylated p16 INK4a genes to: (1) evaluate its potential as a therapeutic agent and (2) investigate its mechanism of action. Demethylation of the p16 INK4a gene and expression of the p16 INK4a protein were observed using higher doses (10 −6–10 −7 M) of drug while growth inhibition at lower doses (IC 50=2×10 −8–4×10 −8 M) was associated with RB dephosphorylation and increased expression of p21 WAF1, but not with induction of p16 INK4a, or apoptosis. Kinetic experiments demonstrated that RB dephosphorylation and the increase of p21 WAF1 preceded the induction of p16 INK4a. The drug induced cell cycle arrest at the G1 and G2/M phases. Antisense experiments demonstrated that the G1 arrest was mediated by transcriptional induction of p21 WAF1. In addition to these observed effects on cell cycle regulatory proteins, decitabine also increased phosphorylation of p38 MAP kinase. The G2/M arrest was inhibited by the p38 MAP kinase inhibitor SB203580, indicating that activation of p38 MAP kinase pathway was required for G2/M arrest. Thus, decitabine inhibited growth by inducing cell cycle arrest at the G1 phase mediated by p21 WAF1 and the G2/M phase through activation of the p38 MAP kinase pathway.

Links between Tumor Suppressors: p53 Is Required for TGF-β Gene Responses by Cooperating with Smads

The p53 tumor suppressor belongs to a family of proteins that sense multiple cellular inputs to regulate cell proliferation, apoptosis, and differentiation. Whether and how these functions of p53 intersect with the activity of extracellular growth factors is not understood. Here, we report that key cellular responses to TGF-β signals rely on p53 family members. During Xenopus embryonic development, p53 promotes the activation of multiple TGF-β target genes. Moreover, mesoderm differentiation is inhibited in p53-depleted embryos. In mammalian cells, the full transcriptional activation of the CDK inhibitor p21 WAF1 by TGF-β requires p53. p53-deficient cells display an impaired cytostatic response to TGF-β signals. Smad and p53 protein complexes converge on separate cis binding elements on a target promoter and synergistically activate TGF-β induced transcription. p53 can physically interact in vivo with Smad2 in a TGF-β-dependent fashion. The results unveil a previously unrecognized link between two primary tumor suppressor pathways in vertebrates.

TPA activates p21 WAF-1 promoter in human T-cells through its second most upstream Sp1 site

The p21 WAF-1 promoter contains binding sites for a number of transcription factors which mediate its activation by a variety of external signals. Moreover, it has been reported that the transcription factors involved in p21 WAF-1 activation by certain signaling factors, like the phorbol ester TPA, may vary in different cell types. We were interested in elucidating the mechanism of p21 WAF-1 activation by TPA in human T-cells, since this activation could explain the antagonistic effect of PKC on apoptosis induction in these cells noted in our previous studies. Using the Jurkat human T-cells we found that TPA activated p21 WAF-1 expression by a PKC-dependent mechanism and that out of six Sp1 binding sites residing in its promoter the second most upstream one was critically essential for this activation. Since p21 WAF-1 is known to inhibit the onset of apoptosis, its PKC-dependent activation may likely account for the PKC antagonistic effect on apoptosis induction in these cells.

Overexpression of cyclin D1 inhibits TNF-induced growth arrest.

Although activated macrophages destroy cancer cells more effectively than normal cells, the facility to escape activated macrophages is a characteristic of tumor cells. One of the mechanisms responsible for the specific killing of tumor cells by macrophages is the production of the cytokine tumor necrosis factor alpha (TNF). Therefore, resistance to TNF may provide such cancer cells a selective advantage against host elimination. In the present work we explore the possibility that cyclin D1 overrides the cytostatic effect of TNF. We show that TNF induces p21(waf1) protein in malignant melanoma A375 cells and its binding to CDK2/4 and 6 proteins, and thereby inhibiting the activity of these complexes. This inhibition leads the cells to a G1 arrest. Overexpression of cyclin D1 in these cells makes them insensitive to TNF treatment with the recovery of CDK activity, however, is unable to overcome the inhibitory action of etoposide blocking the cells on G2/M. The bypass of TNF-induced G1 arrest seems to be related to the increase in the stability of cyclin D bound CDK complexes, increasing the total amount of CDK2/4 and 6 complexes and leading to a functional down titration of the p21(waf1) molecules. In these conditions the TNF-induced increase of p21(waf1) is not sufficient to inhibit the high amount of cyclin D-bound complexes. This hypothesis is supported by the fact that a reduction in the levels of p21(waf1) protein, induced by the expression of a mRNA antisense against p21(waf1), is also able to bypass of TNF-induced arrest. Our results confirm that p21(waf1) has an essential role in TNF-induced arrest and that the deregulation of cyclin D1 may be one of the mechanisms to escape physiological signals to restrict tumoral growth.

Cell-cycle arrest in Jurkat leukaemic cells: a possible role for docosahexaenoic acid.

Docosahexaenoic acid (DHA) is known to have anti-cancer activities by mechanisms that are not well understood. In the present study, we test one possible pathway for DHA action in Jurkat leukaemic cells. Low doses of DHA (10 microM) are shown to induce cell-cycle arrest, whereas higher doses are cytotoxic. However, when cells that were pre-treated with 10 microM DHA are given an additional 10 microM DHA dose, cell viability rapidly decreases. Immunoblotting reveals that repeated low doses of DHA results in activation of caspase 3, implying induction of apoptosis. DHA (10 microM) is shown to increase ceramide levels after 6 h of incubation and, after 24 h, the cells appear to be arrested in S phase. With DHA, the amount of phosphorylated retinoblastoma protein (pRb) decreases significantly. Western blot analysis also shows that DHA greatly reduces the level of cyclin A, while increasing the level of p21 WAF1, a cellular inhibitor of cyclin A/cyclin-dependent kinase 2 (cdk2) activity. Furthermore, the observed DHA-induced doubling of the ratio of hypophosphorylated pRb (hypo-pRb) to total pRb is inhibited by tautomycin and phosphatidic acid (PA), known inhibitors of protein phosphatase 1 (PP1), and by the PP2 inhibitor okadaic acid. The present study demonstrates one possible connected pathway for DHA action. By this pathway, low doses of DHA increase ceramide levels, which leads to inhibition of cdk2 activity and stimulation of PP1 and PP2A. The net effect of cdk2 inhibition and protein phosphatase activation is an inhibition of pRb phosphorylation, consequently arresting Jurkat cell growth.

Mutant p53 can delay growth arrest and loss of CDK2 activity in senescing human fibroblasts without reducing p21 WAF1 expression

Functional wild-type p53 is required for human diploid fibroblasts (HDF) to enter an irreversible growth arrest known as replicative senescence. Experimentally, abrogation of p53 function by expression of human papillomavirus type 16 E6 or disruption of a key downstream effector p21 by homologous recombination both extended HDF life span. However, although sufficient to extend life span, p21 down-regulation is not necessary, because expression of a dominant-negative mutant p53 (143 ala) extends life span without apparently decreasing p21 expression. Given the importance of p53 in cellular senescence and the general assumption that p21 may be the sole mediator of its action in this process, we have investigated how abrogation of p53 function can overcome senescence without lowering expression of p21. We have found up-regulated levels of the cyclin-dependent kinase 2 (cdk2) protein in HDF expressing 143 ala mutant p53 as compared to senescent controls, together with an increase in p21-free cdk2 which, in conjunction with cyclin E, is able to form an active kinase which can phosphorylate the retinoblastoma protein. However, forced overexpression of cdk2 in near-senescent HDF failed to restore cdk2-associated kinase activity. Our data suggest that p53-mediated senescence depends on factor(s) other than p21 which modulate formation of cyclin E-cdk2 complexes.

P21 waf1/cip1 is down-regulated in conjunction with up-regulation of c-Fos in the lymphocytes of rheumatoid arthritis patients

Features characteristic to rheumatoid arthritis (RA) including synovial overgrowth and joint destruction are experimentally produced by augmenting c- fos gene expression. We show that cyclin dependent kinase inhibitor p21 waf1/ cip1 , that inhibits cell proliferation, is down-regulated in conjunction with up-regulation of c- fos in the lymphocytes of patients with RA. As to the mechanism of down-regulation of p21 waf1/ cip1 gene expression, transfection studies in U937 cells showed that c- fos down-regulated phosphorylation and dimerization of signal transducers and activators of transcription (STAT) 1, thereby inhibiting interferon γ-induced transactivation of p21 waf1/ cip1 . Phosphorylation of STAT1 was indeed decreased in the lymphocytes of patients with RA. Thus, under overexpression of c- fos gene, c-Fos inactivates STAT1 to down-regulate p21 waf1/ cip1 gene expression in the lymphocytes of patients with RA, and in this way may enhance proliferation of lymphocytes.

Detection of cell-cycle regulators in failed arteriovenous fistulas for haemodialysis.

Chronic renal failure (CRF) patients rely on arteriovenous fistulas (AVFs) for haemodialysis vascular access. Intimal hyperplastic stenoses result in failure of AVFs and frequent intervention is required to maintain vascular access. The extent of intimal hyperplasia depends on the interplay between cyclins and cyclin-dependent kinases (e.g. cdk2), positively regulating cell-cycle progression. cdk activity is negatively modulated by the interaction with cdk inhibitory proteins, such as p21(Waf1) and p27(Kip1). Little is known about the expression of these proteins in the development of intimal hyperplasia in AVFs. p21(Waf1), p27(Kip1), cdk2 and Proliferating Cell Nuclear Antigen immunoreactivity was determined in 18 failed AVFs from 16 CRF patients and 10 non-diseased vessels (five arteries and five veins). The percentage of p21(Waf1)-positive cells was significantly lower in AVFs (3+/-1%), compared with normal veins and arteries (62+/-4 and 63+/-4%, respectively; P<0.001). cdk2-positive cells were significantly higher in AVFs (40.7+/-3.7%) than in normal veins and arteries (2+/-1 and 0+/-0%, respectively; P<0.001). Although no difference in p27(Kip1) immunoreactivity was found between AVFs (37+/-17%) and veins (23+/-8%; P=0.208), it was lower in healthy arteries (17+/-11%; P=0.037). The data suggest that in failed AVFs, p21(Waf1), but not p27(Kip1), is related to intimal hyperplasia. This is the first report to show involvement of cell-cycle regulators in AVF-related human intimal hyperplasia.

P53 (codon 72) and P21 (codon 31) polymorphisms alter in vivo mRNA expression of p21

p21 ( Waf1/ Cip1) is a downstream target of p53. We evaluated the association between p21 polymorphism (codon 31), p53 polymorphism (codon 72) and their corresponding in vivo mRNA expression. In this study, p21 and p53 genetic polymorphisms (using standard PCR-RFLP techniques) and p21 and p53 gene expressions (using a radiolabelled ribonuclease protection assay (RPA) technique) were evaluated in the peripheral leukocytes of 84 individuals (63 with lung cancer). Log-transformed values of mRNA expression by RPA, which approximated a normal distribution, were analyzed. p53 genotypes did not correlate with p53 mRNA log-expression ( P>0.05 for all comparisons), but the Pro allele variants of p53 were associated with a significant decrease in mRNA log-expression of its downstream target, p21. The variant Arg allele of p21 was also associated with a significant decrease in p21 mRNA log-expression. When individuals with at least one variant allele of both p53 and p21 (double-variants) were compared with all other genotype groups, these double-variants had significantly lower log-expression of p21 ( P<0.005 by both t-tests (crude) and linear regression analyses (adjusted)). This is translated into an approximate 48% reduction in the geometric mean of the mRNA expression of the double-variants, when compared with all other groups. Results were consistent in both patients with lung cancer ( n=63) and in normal controls ( n=21). In conclusion, the presence of a p53 Pro allele and/or p21 Arg allele is associated with lower downstream target gene expression of p21.

Flow cytometry and quantitative immunohistochemical study of cell cycle regulation proteins in invasive breast carcinoma: prognostic significance.

Between January 11, 1991 and January 8, 1992, 104 patients with previously untreated, invasive, primitive breast carcinoma were admitted to the authors' hospital. For each patient, flow cytometry DNA analyses on frozen samples and on immunohistochemical staining were performed, including Ki-67, cyclin A, p53, and p21(waf1) (p21), with assessment of the percentages of positive nuclei were assessed. Correlations with classic clinicopathologic data and survival (overall, metastasis free, or recurrence free) and a multivariate analysis were performed. After a multivariate analysis according to a Cox model that was stratified by age, tumor size, tumor grade, lymph node status, and receptor status, among the factors studied, the presence of p21 was the unique remaining prognostic factor for patients with invasive breast carcinoma. Because of the lack of a correlation between p21 and proliferative factors (Ki-67, S-phase, and cyclin A), the authors combined p21 with those markers and found that, for the different combinations, after statistical analysis, only p21 combined with S-phase or with cyclin A and lymph node status were salient survival prognostic factors. Immunohistochemical study of proteins involved in the cell cycle and assessment of proliferative activity using flow cytometric DNA analysis aided the authors in singling out correlations of cyclin A and S-phase, along with p21, with metastasis free survival and overall survival in patients with invasive breast carcinoma. These promising results will require confirmation in a larger series of patients.

Pyranocoumarins Isolated fromPeucedanum praeruptorumas Differentiation Inducers in Human Leukemic HL-60 Cells

Differentiation therapy for myeloid leukemia offers great potential as a supplement to the current treatment modalities. In the present report, we investigated if the pyranocoumarins, (+/-)-4'- O-acetyl-3'- O-angeloyl- cis-khellactone (or angular pyranocoumarin, APC) isolated from the medicinal plant Peucedanum praeruptorum Dunn, could induce human acute myeloid leukemic HL-60 cells to differentiate and elucidated the molecular mechanism(s) involved. The ability of HL-60 cells to reduce nitroblue tetrazolium (NBT) was significantly increased after APC treatment for 72 h. In these differentiating HL-60 cells, cell surface differentiation markers CD11b (for myeloid cells) and CD14 (for monocytic cells) were detected in 90.3 % and 70.1 % of the cells, respectively. The differentiation inducing effect of APC was time- and dose-dependent. Treatment with 20 microg/mL APC for 72 h inhibited cell growth by 90 % and cell cycle analysis revealed an increase in the proportion of G1 phase cells. In these growth-inhibited cells the expression of the cyclin-dependent kinase inhibitor p27 kip1, but not p21 WAF1, was up-regulated as shown by Western blotting. Differentiation inducing signal pathways were investigated and it was shown that phospho-MEK and phospho-ERK were elevated shortly after the addition of APC. Pre-incubation of the cells with MEK1 inhibitor PD98059 blocked this APC-induced differentiation. Our results suggest that APC are potent inducers of HL-60 cell differentiation along both the myelocytic and monocytic lineages and are potential agents for differentiation-treatment of leukemia.

P53 and a human premature ageing disorder

It has been shown that enhanced levels of p53 activity contribute to reduced cancer susceptibility in mice, however longevity is compromised due to the onset of an early-ageing phenotype. The effects of enhanced levels of p53 in these in mice could therefore have implications for human premature ageing disorders. We examined the DNA damage response of p53 and its target p21 WAF1 to UV and ionising radiation in fibroblasts from patients with the premature ageing disorder Hutchinson-Gilford Progeria (HGP). We report a normal p53 response to these DNA damaging agents suggesting that, in this particular human disorder, the premature ageing phenotype does not arise from an enhanced p53 response.