Extracellular Signal-regulated Kinase 2 (ERK2) Mediates Phosphorylation and Inactivation of Nuclear Interaction Partner of Anaplastic Lymphoma Kinase (NIPA) at G2/M

NIPA is an F-box-like protein that contributes to the timing of mitotic entry. It targets nuclear cyclin B1 for ubiquitination in interphase, whereas in G₂/M phase, NIPA is inactivated by phosphorylation to allow for cyclin B1 accumulation, a critical event for proper G₂/M transition. We recently specified three serine residues of NIPA and demonstrated a sequential phosphorylation at G₂/M, where initial Ser-354 and Ser-359 phosphorylation is most crucial for SCF^NIPA inactivation. In this study, we identified ERK2 as the kinase responsible for this critical initial phosphorylation step. Using in vitro kinase assays, we found that both ERK1 and ERK2 phosphorylated NIPA with high efficiency. Mutation of either Ser-354 or Ser-359 abolished ERK-dependent NIPA phosphorylation. Pharmacologic inhibition of ERK1/2 in cell lines resulted in decreased NIPA phosphorylation at G₂/M. By combining cell cycle synchronization with stable expression of shRNA targeting either ERK1 or ERK2, we showed that ERK2 but not ERK1 mediated NIPA inactivation at G₂/M. ERK2 knockdown led to a delay at the G₂/M transition, a phenotype also observed in cells expressing a phospho-deficient mutant of NIPA. Thus, our data add to the recently described divergent functions of ERK1 and ERK2 in cell cycle regulation, which may be due in part to the differential ability of these kinases to phosphorylate and inactivate NIPA at G₂/M.     

Analysis of radiation‐induced changes to human melanoma cultures using a mathematical model

Objectives: Tumour cells respond to ionizing radiation by cycle arrest, cell death or repair and possible regrowth. We have developed a dynamic mathematical model of the cell cycle to incorporate transition probabilities for entry into DNA replication and mitosis. In this study, we used the model to analyse effects of radiation on cultures of five human melanoma cell lines. Materials and methods: Cell lines were irradiated (9 Gy) prior to further culture and harvesting at multiple points up to 96 h later. Cells were fixed, stained with propidium iodide and analysed for G₁‐, S‐ and G₂/M‐phase cells by flow cytometry. Data for all time points were fitted to a mathematical model. To provide unique solutions, cultures were grown in the presence and absence of the mitotic poison paclitaxel, added to prevent cell division. Results: The model demonstrated that irradiation at 9 Gy induced G₂‐phase arrest in all lines for at least 96 h. Two cell lines with wild‐type p53 status additionally exhibited G₁‐phase arrest with recovery over 15 h, as well as evidence of cell loss. Resumption of cycling of surviving cells, as indicated by increases in G₁/S and G₂/M‐phase transitions, was broadly comparable with results of clonogenic assays. Conclusions: The results, combined with existing data from clonogenic survival assays, support the hypothesis that a dominant effect of radiation in these melanoma lines is the induction of long‐term cell cycle arrest.     

Serine palmitoyltransferase inhibitor myriocin induces growth inhibition of B16F10 melanoma cells through G(2) /M phase arrest

Objectives: Melanoma is the most aggressive form of skin cancer, and it resists chemotherapy. Candidate drugs for effective anti‐cancer treatment have been sought from natural resources. Here, we have investigated anti‐proliferative activity of myriocin, serine palmitoyltransferase inhibitor, in the de novo sphingolipid pathway, and its mechanism in B16F10 melanoma cells. Material and methods: We assessed cell population growth by measuring cell numbers, DNA synthesis, cell cycle progression, and expression of cell cycle regulatory proteins. Ceramide, sphingomyelin, sphingosine and sphingosine‐1‐phosphate levels were analysed by HPLC. Results: Myriocin inhibited proliferation of melanoma cells and induced cell cycle arrest in the G₂/M phase. Expressions of cdc25C, cyclin B1 and cdc2 were decreased in the cells after exposure to myriocin, while expression of p53 and p21^waf1/cip1 was increased. Levels of ceramide, sphingomyelin, sphingosine and sphingosine‐1‐phosphate in myriocin‐treated cells after 24 h were reduced by approximately 86%, 57%, 75% and 38%, respectively, compared to levels in control cells. Conclusions: Our results suggest that inhibition of sphingolipid synthesis by myriocin in melanoma cells may inhibit expression of cdc25C or activate expression of p53 and p21^waf1/cip1, followed by inhibition of cyclin B1 and cdc2, resulting in G₂/M arrest of the cell cycle and cell population growth inhibition. Thus, modulation of sphingolipid metabolism by myriocin may be a potential target of mechanism‐based therapy for this type of skin cancer.     

Cell cycle perturbation by sodium butyrate in tumorigenic and non-tumorigenic human urothelial cell lines assessed by flow cytometric bromodeoxyuridine/DNA analysis

The effect of sodium butyrate on cell proliferation was studied in eight human urothelial cell lines differing in transformation grade (TGr): Hu 1752 (mortal, TGr I); HCV29 (immortal and tumorigenic, TGr II); HCV29T, T24, T24A, T24B, Hu 961A and Hu 1703He (tumorigenic, TGr III). In all cell lines, except Hu 1752, addition of 4 mm sodium butyrate at 18 h after replating resulted in a significantly decreased population of adherent cells after a further 24–48 h. This might partially be explained by detachment of cells, probably mainly S phase cells, from the substrate in the lines HCV 29, HCV29T, Hu 961A and Hu 1703He. Flow cytometric DNA analysis of the adherent cell population showed that all TGr II and III urothelial cell lines were DNA aneuploid, and that butyrate perturbed the cell cycle distribution in these cell lines, mainly by a decrease of the S phase fraction. Flow cytometric bromodeoxyuridine (BrdUrd)/DNA analysis of continuously BrdUrd labelled cultures, using a ‘washless’ BrdUrd/DNA staining technique, showed that butyrate inhibited the G_0/1-S phase transition, indicated by a delayed depletion of BrdUrd negative G_0/1 cells in the cell lines HCV29, HCV29T, T24B, Hu 961A and Hu 1703He. BrdUrd/DNA analysis further showed that butyrate inhibited the G₂M-G_0/1 phase transition, indicated by a pronounced accumulation of BrdUrd positive G₂M cells in the cell lines HCV29T, T24B, Hu 961A and Hu 1703He. Microscopy of HCV29T and Hu 961A cells indicated that this block did not occur in mitosis. The parental cell line T 24 and the cell line T 24 A did not show an accumulation of BrdUrd negative G_0/1 cells or BrdUrd positive G₂M cells like that occurring in the derived cell line T 24B.     

Skp2 regulates G2/M progression in a p53-dependent manner

Targeted proteasomal degradation mediated by E3 ubiquitin ligases controls cell cycle progression, and alterations in their activities likely contribute to malignant cell proliferation. S phase kinase-associated protein 2 (Skp2) is the F-box component of an E3 ubiquitin ligase complex that targets p27^Kip1 and cyclin E1 to the proteasome. In human melanoma, Skp2 is highly expressed, regulated by mutant B-RAF, and required for cell growth. We show that Skp2 depletion in melanoma cells resulted in a tetraploid cell cycle arrest. Surprisingly, co-knockdown of p27^Kip1 or cyclin E1 failed to prevent the tetraploid arrest induced by Skp2 knockdown. Enhanced Aurora A phosphorylation and repression of G2/M regulators cyclin B1, cyclin-dependent kinase 1, and cyclin A indicated a G2/early M phase arrest in Skp2-depleted cells. Furthermore, expression of nuclear localized cyclin B1 prevented tetraploid accumulation after Skp2 knockdown. The p53 status is most frequently wild type in melanoma, and the tetraploid arrest and down-regulation of G2/M regulatory genes were strongly dependent on wild-type p53 expression. In mutant p53 melanoma lines, Skp2 depletion did not induce cell cycle arrest despite up-regulation of p27^Kip1. These data indicate that elevated Skp2 expression may overcome p53-dependent cell cycle checkpoints in melanoma cells and highlight Skp2 actions that are independent of p27^Kip1 degradation.     

Alpha-santalol,a chemopreventive agent against skin cancer,causes G<Subscript>2</Subscript>/M cell cycle arrest in both p53-mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells

Background

α-Santalol, an active component of sandalwood oil, has shown chemopreventive effects on skin cancer in different murine models. However, effects of α-santalol on cell cycle have not been studied. Thus, the objective of this study was to investigate effects of α-santalol on cell cycle progression in both p53 mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells to elucidate the mechanism(s) of action.

Methods

MTT assay was used to determine cell viability in A431 cells and UACC-62; fluorescence-activated cell sorting (FACS) analysis of propidium iodide staining was used for determining cell cycle distribution in A431 cells and UACC-62 cells; immunoblotting was used for determining the expression of various proteins and protein complexes involved in the cell cycle progression; siRNA were used to knockdown of p21 or p53 in A431 and UACC-62 cells and immunofluorescence microscopy was used to investigate microtubules in UACC-62 cells.

Results

α-Santalol at 50-100 μM decreased cell viability from 24 h treatment and α-santalol at 50 μM-75 μM induced G₂/M phase cell cycle arrest from 6 h treatment in both A431 and UACC-62 cells. α-Santalol altered expressions of cell cycle proteins such as cyclin A, cyclin B1, Cdc2, Cdc25c, p-Cdc25c and Cdk2. All of these proteins are critical for G₂/M transition. α-Santalol treatment up-regulated the expression of p21 and suppressed expressions of mutated p53 in A431 cells; whereas, α-santalol treatment increased expressions of wild-type p53 in UACC-62 cells. Knockdown of p21 in A431 cells, knockdown of p21 and p53 in UACC-62 cells did not affect cell cycle arrest caused by α-santalol. Furthermore, α-santalol caused depolymerization of microtubules similar to vinblastine in UACC-62 cells.

Conclusions

This study for the first time identifies effects of α-santalol in G₂/M phase arrest and describes detailed mechanisms of G₂/M phase arrest by this agent, which might be contributing to its overall cancer preventive efficacy in various mouse skin cancer models.

     

Surfactin Induces Apoptosis and G<Subscript>2</Subscript>/M Arrest in Human Breast Cancer MCF-7 Cells Through Cell Cycle Factor Regulation

Surfactin, purified from Bacillus subtilis natto TK-1, inhibited proliferation of human breast cancer MCF-7 cells in a dose- and time-dependent manner, with IC₅₀ at 24, 48, and 72 h of 82.6, 27.3, and 14.8 μM, respectively. Surfactin-induced cell death was considered to be apoptotic by observing the typical apoptotic morphological change by acridine orange/ethidium bromide staining and Transferase-mediated dUTP Nick End-labeling assay. [Ca²⁺]i measurement revealed that surfactin induced a sustained increase in concentration of intracellular [Ca²⁺]i. Flow cytometric analysis also demonstrated that surfactin caused time-dependent apoptosis of MCF-7 cells through cell arrest at G₂/M phase. Western blot revealed that surfactin induced accumulation of the tumor suppressor p53 and cyclin kinase inhibitor p21^waf1/cip1, and inhibited the activity of the G₂-specific kinase, cyclin B1/p34^cdc2. Based on our findings, surfactin inhibited proliferation in MCF-7 cells by inducing apoptosis and the elevation of [Ca²⁺]i may play an important role in the apoptosis. The mechanism which surfactin caused G₂/M arrest seems to be through cell cycle factor regulation.     

Murine coronavirus replication induces cell cycle arrest in G0/G1 phase

Mouse hepatitis virus (MHV) replication in actively growing DBT and 17Cl-1 cells resulted in the inhibition of host cellular DNA synthesis and the accumulation of infected cells in the G₀/G₁ phase of the cell cycle. UV-irradiated MHV failed to inhibit host cellular DNA synthesis. MHV infection in quiescent 17Cl-1 cells that had been synchronized in the G₀ phase by serum deprivation prevented infected cells from entering the S phase after serum stimulation. MHV replication inhibited hyperphosphorylation of the retinoblastoma protein (pRb), the event that is necessary for cell cycle progression through late G₁ and into the S phase. While the amounts of the cellular cyclin-dependent kinase (Cdk) inhibitors p21^Cip1, p27^Kip1, and p16^INK4a did not change in infected cells, MHV infection in asynchronous cultures induced a clear reduction in the amounts of Cdk4 and G₁ cyclins (cyclins D1, D2, D3, and E) in both DBT and 17Cl-1 cells and a reduction in Cdk6 levels in 17Cl-1 cells. Infection also resulted in a decrease in Cdk2 activity in both cell lines. MHV infection in quiescent 17Cl-1 cells prevented normal increases in Cdk4, Cdk6, cyclin D1, and cyclin D3 levels after serum stimulation. The amounts of cyclin D2 and cyclin E were not increased significantly after serum stimulation in mock-infected cells, whereas they were decreased in MHV-infected cells, suggesting the possibility that MHV infection may induce cyclin D2 and cyclin E degradation. Our data suggested that a reduction in the amounts of G₁ cyclin-Cdk complexes in MHV-infected cells led to a reduction in Cdk activities and insufficient hyperphosphorylation of pRb, resulting in inhibition of the cell cycle in the G₀/G₁ phase.     

Cellular radiation effects and hyperthermia: Cytokinetic investigations with stationary phase yeast cells

Summary Wild type diploid yeast, Saccharomyces cerevisiae strain 211, was subjected to 250 kV X-rays or 50° C heat treatment for 30 min or to a combination of both. X-ray exposure took place either in air or in nitrogen. Cell number, percentage of budding cells and cell cycle progression was followed for up to 12 h post irradiation. The distribution of cell cycle stages was determined by flow cytofluorometry. All treatments cause a retardation of cell division rate. Hyperthermia leads mainly to a lengthening of G₁, whereas X-rays arrest the cells reversibly in G₂. The effect of the combined treatment appears to be merely additive. No selective action of hyperthermia on hypoxic cells was found.Dedicated to Prof. Dr. A. Schraub on the occasion of his 70th birthday     

The mechanisms of nadroparin‐mediated inhibition of proliferation of two human lung cancer cell lines

Objectives

Clinical data suggest that heparin treatment improves survival of lung cancer patients, but the mechanisms involved are not fully understood. We investigated whether low molecular weight heparin nadroparin, directly affects lung cancer cell population growth in conventionally cultured cell lines.

Materials and methods

A549 and CALU1 cells’ viability was assessed by MTT and trypan blue exclusion assays. Cell proliferation was assessed using 5‐bromo‐2‐deoxyuridine incorporation. Apoptosis and cell‐cycle distribution were analysed by flow cytometry; cyclin B1, Cdk1, p‐Cdk1 Cdc25C, p‐Cdc25C and p21 expressions were analysed by western blotting. mRNA levels were analysed by real time RT‐PCR.

Results

Nadroparin inhibited cell proliferation by 30% in both cell lines; it affected the cell cycle in A549, but not in CALU‐1 cells, inducing arrest in the G₂/M phase. Nadroparin in A549 culture inhibited cyclin B1, Cdk1, Cdc25C and p‐Cdc25C, while levels of p‐Cdk1 were elevated; p21 expression was not altered. Dalteparin caused a similar reduction in A549 cell population growth; however, it did not alter cyclin B1 expression as expected, based on previous reports. Fondaparinux caused minimal inhibition of A549 cell population growth and no effect on either cell cycle or cyclin B1 expression.

Conclusions

Nadroparin inhibited proliferation of A549 cells by inducing G₂/M phase cell‐cycle arrest that was dependent on the Cdc25C pathway, whereas CALU‐1 cell proliferation was halted by as yet not elucidated modes.     

The differentiation of T-lymphocytes. III. The behaviour of subpopulations of mouse thymus cells in short-term cell culture.

Seven human cultured lymphoblastoid cell lines (CLL) were divided into two major groups based on studies of their cell cycle characteristics and surface Ig. CLL I (lines CL, MW, HH and TM) had generation times ranging from 25–40 hr, S phase times of 10–12 hr, G₂ + M times of 6–8 hr, and demonstrated sharp differences between the percentage of SIg(+) cells in different phases of the cell cycle. Line TM was particularly discordant with the highest percentage of SIg(+) cells in G₂ + M. CLL II (lines PS, JR and HT) demonstrated generation times ranging from 18–21 hr, S phase times of 7–10 hr and G₂ + M phase times of 2 hr. In this second group, two of the three CLLs had no differences between cells taken from different points of the cell cycle. DNA synthesis and cell density could not be correlated with either of the above major parameters, i.e. cell cycle times or SIg expression. The results suggest that human CLLs fall into subgroups in which specific patterns of cellular and immune functions may predominate.     

The pan-ErbB receptor tyrosine kinase inhibitor canertinib promotes apoptosis of malignant melanoma in vitro and displays anti-tumor activity in vivo

The ErbB receptor family has been suggested to constitute a therapeutic target for tumor-specific treatment of malignant melanoma. Here we investigate the effect of the pan-ErbB tyrosine kinase inhibitor canertinib on cell growth and survival in human melanoma cells in vitro and in vivo. Canertinib significantly inhibited growth of cultured melanoma cells, RaH3 and RaH5, in a dose-dependent manner as determined by cell counting. Half-maximum growth inhibitory dose (IC₅₀) was approximately 0.8 μM and by 5 μM both cell lines were completely growth-arrested within 72 h of treatment. Incubation of exponentially growing RaH3 and RaH5 with 1 μM canertinib accumulated the cells in the G₁-phase of the cell cycle within 24 h of treatment without induction of apoptosis as determined by flow cytometry. Immunoblot analysis showed that 1 μM canertinib inhibited ErbB1-3 receptor phosphorylation with a concomitant decrease of Akt-, Erk1/2- and Stat3 activity in both cell lines. In contrast to the cytostatic effect observed at doses ?5 μM canertinib, higher concentrations induced apoptosis as demonstrated by the Annexin V method and Western blot analysis of PARP cleavage. Furthermore, canertinib significantly inhibited growth of RaH3 and RaH5 melanoma xenografts in nude mice. Pharmacological targeting of the ErbB receptors may prove successful in the treatment of patients with metastatic melanoma.     

Threshold expression of cyclin E but not D type cyclins characterizes normal and tumour cells entering S phase

Complexes of cyclin-dependent kinases (cdk) and their partner cyclins drive the cell through the cell cycle, each such complex phosphorylating a distinct set of proteins at a particular check-point or phase of the cycle. Immunocytochemical detection of cyclins combined with measurement of cellular DNA content by flow cytometry makes it possible to relate expression of each of these proteins with the actual cell cycle position, without the necessity of cell synchronization. In the present study, we have investigated expression of E and D type cyclins in G₁ cells and in cells entering S phase, in eight different human hematopoietic and solid tumour cell lines (two leukaemias, a lymphoma, three breast carcinomas, a colon carcinoma and a bladder transitional cell carcinoma) during their exponential phase of growth, as well as in normal mitogen stimulated lymphocytes. In all the cell types studied, the average level of D type cyclin expression was invariable throughout the cell cycle. A great intercellular variability, in particular of the G₁ cell subpopulations, and the presence of a large fraction of G₁, S and G₂+ M cells that were cyclin D negative (20–40% in tumour cell lines and about 80% among lymphocytes), were other characteristic features of D type cyclin expression. In contrast to D type cyclins, the expression of cyclin E was discontinuous during the cycle, peaking at the time of cell entrance to S. Also, a well defined threshold in expression of cyclin E characterized cells that were entering S phase, and virtually no cyclin E negative cells were seen during the early portion of S phase. The data indicate that while cell entrance to S phase is unrelated to expression of D type cyclins (at the time of entrance), accumulation of cyclin E up to critical level is a prerequisite for initiation of DNA replication. The great intercellular variability in expression of D type cyclins and their invariant average level across the cell cycle suggest that these cyclins, in addition to their acknowledged function in promoting cell progression through mid- to late-G₁ may have other role(s), related or unrelated to the cell cycle progression. The presence of a large number of D type cyclin negative cells in all phases of the cycle suggests that during exponential growth the cells may not express this protein and yet may traverse the cycle, including G₁ phase.     

Mitofusin 1 is degraded at G<Subscript>2</Subscript>/M phase through ubiquitylation by MARCH5

Background

Mitochondria exhibit a dynamic morphology in cells and their biogenesis and function are integrated with the nuclear cell cycle. In mitotic cells, the filamentous network structure of mitochondria takes on a fragmented form. To date, however, whether mitochondrial fusion activity is regulated in mitosis has yet to be elucidated.

Findings

Here, we report that mitochondria were found to be fragmented in G₂ phase prior to mitotic entry. Mitofusin 1 (Mfn1), a mitochondrial fusion protein, interacted with cyclin B1, and their interactions became stronger in G₂/M phase. In addition, MARCH5, a mitochondrial E3 ubiquitin ligase, reduced Mfn1 levels and the MARCH5-mediated Mfn1 ubiquitylation were enhanced in G₂/M phase.

Conclusions

Mfn1 is degraded through the MARCH5-mediated ubiquitylation in G₂/M phase and the cell cycle-dependent degradation of Mfn1 could be facilitated by interaction with cyclin B1/Cdk1 complexes.

     

BH3-mimetic gossypol-induced autophagic cell death in mutant BRAF melanoma cells with high expression of p21

Aims

The aim of the present study was to identify the potential therapeutic effects of BH3-mimetic gossypol on melanoma cells with acquired resistance to BRAF inhibitors.

Main methods

The IC₅₀ values of gossypol were determined using MTT assays in three melanoma cell lines with different resistances to BRAF inhibitor. The effects of gossypol on three melanoma cell lines were further examined by immunoblotting analysis, cell cycle analysis, flow cytometric apoptotic assay and autophagy assay. The functional role of autophagy in gossypol-induced growth inhibition was investigated using siRNA-mediated knockdown of Beclin-1.

Key findings

Gossypol retained its efficacy in BRAF-V600E melanoma clones with acquired resistance to BRAF inhibitors through a mechanism independent of MEK–ERK inhibition. Gossypol caused G₂/M arrest in both BRAF mutant A375P and A375P/Mdr cells with high expression of p21^Cip1, regardless of their drug resistance. Interestingly, we determined that the lack of gossypol-induced mitotic arrest in BRAF-WT-harboring SK-MEL-2 cells was associated with a low level of p21^Cip1 expression. In addition, gossypol preferentially induced autophagy and apoptosis in the gossypol-sensitive cells and not in the gossypol-resistant SK-MEL-2 cells. In particular, alleviation of autophagy by knockdown of Beclin-1 partially caused a resistance to gossypol-induced cell cycle arrest at G₂/M in BRAF-V600E cells with a concomitant decreased induction of apoptosis.

Significance

Taken together, these results suggest that gossypol may exhibit potential for the treatment of BRAF inhibitor-resistant tumors, but a functional p21^Cip1 is a prerequisite for a positive response to its clinical application.     

Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis

Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A₂ (TxA₂)/TxA₂ receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G₂/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G₂/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA₂/TP promotes MM cell proliferation by reducing cell delay at G₂/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy.     

Cell cycle regulation during growth-dormancy cycles in pea axillary buds

Accumulation patterns of mRNAs corresponding to histones H2A and H4, ribosomal protein genes rpL27 and rpL34, MAP kinase, cdc2 kinase and cyclin B were analyzed during growth-dormancy cycles in pea (Pisum sativum cv. Alaska) axillary buds. The level of each of these mRNAs was low in dormant buds on intact plants, increased when buds were stimulated to grow by decapitating the terminal bud, decreased when buds ceased growing and became dormant, and then increased when buds began to grow again. Flow cytometry was used to determine nuclear DNA content during these developmental transitions. Dormant buds contain G₁ and G₂ nuclei (about 3:1 ratio), but only low levels of S phase nuclei. It is hypothesized that cells in dormant buds are arrested at three points in the cell cycle, in mid-G₁, at the G₁/S boundary and near the S/G₂ boundary. Based on the accumulation of histone H2A and H4 mRNAs, which are markers for S phase, cells arrested at the G₁/S boundary enter S within one hour of decaptitation. The presence of a cell population arrested in mid-G₁ is indicated by a second peak of histone mRNA accumulation 6 h after the first peak. Based on the accumulation of cyclin B mRNA, a marker for late G₂ and mitosis, cells arrested at G₁/S begin to divide between 12 and 18 h after decapitation. A small increase in the level of cyclin B mRNA at 6 h after decapitation may represent mitosis of the cells that had been arrested near the S/G₂ boundary. Accumulation of MAP kinase, cdc2 kinase, rpL27 and rpL34 mRNAs are correlated with cell proliferation but not with a particular phase of the cell cycle.     

Resveratrol induces cell‐cycle disruption and apoptosis in chemoresistant B16 melanoma

Resveratrol, a naturally occurring polyphenol, has been shown to possess chemopreventive activities. In this study, we show that resveratrol (0–500 µM) inhibits the growth of a doxorubicin‐resistant B16 melanoma cell subline (B16/DOX) (IC₅₀ = 25 µM after 72 h, P < 0.05). This was accomplished by imposing an artificial checkpoint at the G₁–S phase transition, as demonstrated by cell‐cycle analysis and down‐regulation of cyclin D1/cdk4 and increased of p53 expression level. The G₁‐phase arrest of cell cycle in resveratrol‐treated (10–100 µM) B16/DOX cells was followed by the induction of apoptosis, which was revealed by pyknotic nuclei and fragmented DNA. Resveratrol also potentiated at subtoxic dose (25 µM for 24 h) doxorubicin cytotoxicity in the chemoresistant B16 melanoma (P < 0.01). When administered to mice, resveratrol (12.5 mg/kg) reduced the growth of an established B16/DOX melanoma and prolonged survival (32% compared to untreated mice). All these data support a potential use of resveratrol alone or in combination with other chemotherapeutic agents in the management of chemoresistant tumors. J. Cell. Biochem. 110: 893–902, 2010. © 2010 Wiley‐Liss, Inc.