BW18, a C-21 steroidal glycoside, exerts an excellent anti-leukemia activity through inducing S phase cell cycle arrest and apoptosis via MAPK pathway in K562 cells.

C-21 steroids displayed the activities of immunosuppressive, anti-inflammatory and anti-virus effects by the reports. However, its antitumor effects and molecular mechanism remain unclear. We previously isolated and identified a C-21 steroidal glycoside (BW18) from the root of Cynanchum atratum Bunge. This study was aimed to assess anti-leukemia activity and its underlying mechanism in K562 cells. MTT assay results showed that BW18 inhibited cell viability and proliferation of K562 cells. We also found that BW18 could induce S phase cell cycle arrest and apoptosis. Furthermore, our results demonstrated that BW18 regulated the expression of apoptosis and cell cycle related proteins. Mechanism investigation revealed that the anti-leukemia activity of BW18 may be mediated through MAPK pathway. These findings indicate that BW18 possesses an excellent anti-leukemia activity via regulating MAPK pathway leading to S phase cell cycle arrest and apoptosis, which suggested BW18 could be as a potential alternative therapeutic agent for CML patients.

Achyranthes aspera root extracts induce human colon cancer cell (COLO-205) death by triggering the mitochondrial apoptosis pathway and S phase cell cycle arrest.

Achyranthes aspera (AA) has been used traditionally for the cure of various disorders. However, the action of root extracts of AA as anticancer agent and its cellular mechanism remain unclear. The aim was to screen the antitumor effect of ethanolic (EAA) and aqueous (AAA) root extracts on the growth of colon cancer COLO-205 cells by testing their cytotoxicity, followed by their effect on clonogenicity, migration, and induction of apoptosis. Mechanisms leading to apoptosis and cell cycle arrest were also investigated by expression studies of caspase-9, caspase-3, Bax, Bcl-2, p16, p21, and p27 genes, followed by flow cytometric analysis for cell cycle distribution. Cytotoxicity screening of AA extracts indicated greater cytotoxic activity of AAA extract against COLO-205 cells. A series of events marked by apoptosis revealed loss of cell viability, chromatin condensation, and DNA fragmentation in AAA treated cells to a greater extent. The mRNA expression levels of caspase-9, caspase-3, Bax, p16, p21, and p27 were markedly increased in the AAA treated cells, along with decreased Bcl-2 expression. The cell cycle arrest at S phase was detected by flow cytometric analysis after treatment with AAA. Overall the study signifies the aqueous extracts as a promising therapeutic candidate against cancer.

DNA topoisomerase II-dependent control of the cell cycle progression in root meristems of Allium cepa.

The catalytic ability of DNA topoisomerases (Topo) to generate short-term DNA breaks allow these enzymes to play crucial functions in managing DNA topology during S-phase replication, transcription, and chromatin-remodelling processes required to achieve commitment for the onset and transition through mitosis. Our experiments on root meristem cells of onion (Allium cepa) were designed to gain insight into the contribution of Topo II to plant-specific progression throughout interphase and mitosis. Irrespective of the position of the cell in interphase, the immunofluorescence of Topo II revealed similar nuclear labelling pattern with well defined signals dispersed in the nucleoplasm and the cortical zone of the nucleolus. Only weak labelling was detected in metaphase and anaphase chromosomes. Experiments with two potent anti-Topo II agents, doxorubicin (DOX, an anthracycline) and a bisdioxopiperazine derivative, ICRF-193, suggest that the inhibition-mediated increase in Topo II immunofluorescence may represent a compensatory mechanism, by which an up-regulated expression of the enzyme tends to counteract the drug-induced loss of indispensable catalytic and relaxation functions. γ-H2AX immunolabelling seems to indicate that both DOX- and ICRF-193-induced alterations in cell cycle progression reflect primarily the activity of the G2/M DNA damage checkpoint. Our findings provide evidence for the plant-specific cell cycle control mechanism induced by Topo II inhibitors under DNA stress conditions.

Pathologic response to short intensified taxane-free neoadjuvant chemotherapy in patients with highly proliferative operable breast cancer.

OBJECTIVES: Breast cancer treatment relies on 3 major phenotypical subtypes, including the triple-negative (TN), HER2-positive, and hormone receptor-positive (estrogen receptor/progesterone receptor) ones. We retrospectively determined the clinical and pathologic response rates to intensified taxane-free neoadjuvant chemotherapy according to these phenotypical classes in a series of patients with highly proliferative operable breast cancer, and examined the patterns of recurrence. METHODS: Patients with early breast cancer with highly proliferative (S-phase fraction >4%) operable tumors of >3 cm received 4 cycles of intensified neoadjuvant chemotherapy with high-dose cyclophosphamide (doxorubicin 70 mg/m d1, cyclophosphamide 700 mg/m d1/d8, and 5 FU 700 mg/m d1-d5) every 3 weeks. RESULTS: Fifty-five patients were included in the analysis. Patients with TN phenotype experienced a high pathologic complete response (pCR) rate to intensified chemotherapy in comparison with patients with HER2-positive and estrogen receptor/progesterone receptor tumors (47%, 0%, and 12%, respectively). Forty percent of patients with TN breast cancer recurred after a median follow-up of nearly 11 years, but only 22% of those achieving a pCR. CONCLUSIONS: A high pCR rate to short intensified neoadjuvant chemotherapy with high-dose cyclophosphamide was achieved in patients with operable highly proliferative TN breast cancer, and pCR was associated with a low rate of recurrence.

Cooperativity between inhibition of cytosolic K+ efflux and AMPK activation during suppression of hypoxia-induced cellular apoptosis.

Cellular potassium homeostasis has recently emerged as a critical regulator of apoptosis in response to variety of stimuli. However, functional hierarchy of this phenomenon in the apoptotic cascade and therefore, its significance as a pathway for intervention is not fully established. Chronic hypoxia, a known threat to cell survival, also modulates cellular potassium homeostasis. In this study, we tested if hypoxia-induced apoptosis in lymphocytes can be prevented by modulating cellular K+ homeostasis. We observed that chronic hypoxia accelerated the rate of apoptosis in resting murine splenocytes concomitant with cytosolic K+ efflux. We tested several modalities including elevated extracellular potassium besides various K+ channel inhibitors to curtail hypoxia-induced K+ efflux and interestingly, established that the supplementation of KCl in extracellular medium was most effective in preventing hypoxia-induced apoptosis in these cells. Subsequent mechanistic dissection of pathways underlying this phenomenon revealed that besides effectively inhibiting hypoxia-induced efflux of K+ ion and its downstream cell-physiological consequences; elevated extracellular KCl modulated steady state levels of cellular ATP and culminated in stabilization of AMPKα with pro-survival consequences. Also, interestingly, global gene expression profiling revealed that KCl supplementation down regulated a distinct p53-regulated cellular sub-network of genes involved in regulation of DNA replication. Additionally, we present experimental evidence for the functional role of AMPK and p53 activation during suppression of hypoxia-induced apoptosis. In conclusion, our study highlights a novel bimodal effect wherein cooperativity between restoration of K+ homeostasis and a sustainable 'metabolic quiescence' induced by AMPK activation appeared indispensible for curtailing hypoxia-induced apoptosis.

Preclinical evaluation of synergistic effect of telomerase-specific oncolytic virotherapy and gemcitabine for human lung cancer.

A phase I dose-escalation study of telomerase-specific oncolytic adenovirus, OBP-301 (Telomelysin), is now under way in the United States to assess feasibility and to characterize its pharmacokinetics in patients with advanced solid tumors. The present preclinical study investigates whether OBP-301 and a chemotherapeutic agent that is commonly used for lung cancer treatment, gemcitabine, are able to enhance antitumor effects in vitro and in vivo. The antitumor effects of OBP-301 infection and gemcitabine were evaluated by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt assay. In vivo antitumor effects of intratumoral injection of OBP-301 in combination with systemic administration of gemcitabine were assessed on nu/nu mice s.c. xenografted with human lung tumors. OBP-301 infection combined with gemcitabine resulted in very potent synergistic cytotoxicity in human lung cancer cells. The three human lung cancer cell lines treated with OBP-301 for 24 hours tended to accumulate in S phase compared with controls. The proportion of cells in S phase increased from 43.85% to 56.41% in H460 cells, from 46.72% to 67.09% in H322 cells, and from 38.22% to 57.67% in H358 cells. Intratumoral injection of OBP-301 combined with systemic administration of gemcitabine showed therapeutic synergism in human lung tumor xenografts. Our data suggest that the combination of OBP-301 and gemcitabine enhances the antitumor effects against human lung cancer. We also found that the synergistic mechanism may be due to OBP-301-mediated cell cycle accumulation in S phase. These results have important implications for the treatment of human lung cancer.

Inhibition of calcium-independent phospholipase A2 suppresses proliferation and tumorigenicity of ovarian carcinoma cells.

PLA2 (phospholipase A2) enzymes play critical roles in membrane phospholipid homoeostasis and in generation of lysophospholipid growth factors. In the present study, we show that the activity of the cytosolic iPLA2 (calcium-independent PLA2), but not that of the calcium-dependent cPLA2 (cytosolic PLA2), is required for growth-factor-independent, autonomous replication of ovarian carcinoma cells. Blocking iPLA2 activity with the pharmacological inhibitor BEL (bromoenol lactone) induces cell cycle arrest in S- and G2/M-phases independently of the status of the p53 tumour suppressor. Inhibition of iPLA2 activity also leads to modest increases in apoptosis of ovarian cancer cells. The S- and G2/M-phase accumulation is accompanied by increased levels of the cell cycle regulators cyclins B and E. Interestingly, the S-phase arrest is released by supplementing the growth factors LPA (lysophosphatidic acid) or EGF (epidermal growth factor). However, inhibition of iPLA2 activity with BEL remains effective in repressing growth-factor- or serum-stimulated proliferation of ovarian cancer cells through G2/M-phase arrest. Down-regulation of iPLA2b expression with lentivirus-mediated RNA interference inhibited cell proliferation in culture and tumorigenicity of ovarian cancer cell lines in nude mice. These results indicate an essential role for iPLA2 in cell cycle progression and tumorigenesis of ovarian carcinoma cells.

Dietary beta-carotene inhibits mammary carcinogenesis in rats depending on dietary alpha-linolenic acid content.

To investigate whether dietary alpha-linolenic acid (ALA) content alters the effect of beta-carotene on mammary carcinogenesis, we conducted a chemically induced mammary tumorigenesis experiment in rats randomly assigned to four nutritional groups (15 rats per group) varying in beta-carotene supplementation and ALA content. Two oil formula-enriched diets (15 %) were used: one with 6 g ALA/kg diet in an essential fatty acids (EFA) ratio of linoleic acid:ALA of 5:1 w/w (EFA 5 diet), the other with 24 g ALA/kg diet in an EFA ratio of 1:1 w/w (EFA 1 diet), both designed with a similar linoleic acid content. beta-Carotene was either added (10 mg/kg diet per d) or not added to these diets. beta-Carotene supplementation led to decreased tumour incidence and tumour growth when added to the EFA 5 diet, whereas it had no effect when added to the EFA 1 diet. The decreased tumour growth did not result from an involvement of lipoperoxidation (tumour malondialdehyde content being similar between the groups) or from an inhibition of tumour cell proliferation (as there was an unchanged S phase fraction in the tumours). We concluded that an adequate content of ALA in the diet is required to allow a protective effect of beta-carotene in mammary carcinogenesis. Whether such an interaction between ALA and beta-carotene influences the risk of breast cancer in women needs to be investigated.

The heat-shock protein Apg-2 binds to the tight junction protein ZO-1 and regulates transcriptional activity of ZONAB.

The tight junction adaptor protein ZO-1 regulates intracellular signaling and cell proliferation. Its Src homology 3 (SH3) domain is required for the regulation of proliferation and binds to the Y-box transcription factor ZO-1-associated nucleic acid binding protein (ZONAB). Binding of ZO-1 to ZONAB results in cytoplasmic sequestration and hence inhibition of ZONAB's transcriptional activity. Here, we identify a new binding partner of the SH3 domain that modulates ZO-1-ZONAB signaling. Expression screening of a cDNA library with a fusion protein containing the SH3 domain yielded a cDNA coding for Apg-2, a member of the heat-shock protein 110 (Hsp 110) subfamily of Hsp70 heat-shock proteins, which is overexpressed in carcinomas. Regulated depletion of Apg-2 in Madin-Darby canine kidney cells inhibits G(1)/S phase progression. Apg-2 coimmunoprecipitates with ZO-1 and partially localizes to intercellular junctions. Junctional recruitment and coimmunoprecipitation with ZO-1 are stimulated by heat shock. Apg-2 competes with ZONAB for binding to the SH3 domain in vitro and regulates ZONAB's transcriptional activity in reporter gene assays. Our data hence support a model in which Apg-2 regulates ZONAB function by competing for binding to the SH3 domain of ZO-1 and suggest that Apg-2 functions as a regulator of ZO-1-ZONAB signaling in epithelial cells in response to cellular stress.

The mathematical modelling of adjuvant chemotherapy scheduling: incorporating the effects of protocol rest phases and pharmacokinetics.

In this paper the modelling objective is to determine the drug alternation time which minimises the formation of resistant tumour cells when delivering two non-cross resistant chemotherapeutics given such drugs cannot be delivered simultaneously and constraints due to pharmacokinetics and protocol rest phases. We initially consider cell cycle phase non-specific models, as investigated by Goldie and Coldman. By extending previous work, these models are generalised to consider chemotherapeutic S-phase specificity. We find with the cell cycle phase non-specific models that once the alternation time of the drugs is reduced below a critical threshold, a substantial improvement in protocol outcome is predicted. Extensive improvements are also observed for the S-phase specific investigation if the drugs can be alternated extremely rapidly. However, this is typically impossible due to pharmacokinetic constraints. Under such circumstances, the most appropriate choice of the alternation time can depend sensitively on the median and variance of the tumour cell cycle time in a complicated manner. For schedulings motivated by Capecitabine protocols, we find that switching the drugs only once, or at most twice, between rest phases gives the most reliable alternation time. The main and novel conclusion of this paper is the modelling prediction that one must be much more specific in the choice of the protocol alternation time if attempting to observe the improvements promised by Goldie and Coldman's alternation hypothesis for the rest phases, pharmacokinetics and delivery mechanisms typically encountered in cell cycle phase specific chemotherapy protocols.

Arginine methylation of MRE11 by PRMT1 is required for DNA damage checkpoint control.

The role of protein arginine methylation in the DNA damage checkpoint response and DNA repair is largely unknown. Herein we show that the MRE11 checkpoint protein is arginine methylated by PRMT1. Mutation of the arginines within MRE11 severely impaired the exonuclease activity of MRE11 but did not influence its ability to form complexes with RAD50 and NBS1. Cells containing hypomethylated MRE11 displayed intra-S-phase DNA damage checkpoint defects that were significantly rescued with the MRE11-RAD50-NBS1 complex. Our results suggest that arginine methylation regulates the activity of MRE11-RAD50-NBS1 complex during the intra-S-phase DNA damage checkpoint response.

Molecular mechanisms of enhanced renal cell division in protection against S-1,2-dichlorovinyl-L-cysteine-induced acute renal failure and death.

Sustained activation of ERK 1/2 by a low dose (15 mg/kg ip) of S-1,2-dichlorovinyl-l-cysteine (DCVC) 72 h before administration of a lethal dose of DCVC (75 mg/kg ip) enhances renal cell division and protects mice against acute renal failure (ARF) and death (autoprotection). The objective of this study was to determine correlation among extent of S-phase DNA synthesis, activation of transcription factors, expression of G(1)/S cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors downstream of ERK 1/2 following DCVC-induced ARF in autoprotection. Administration of the lethal dose alone caused a general downregulation or an unsustainable increase, in transcriptional and posttranscriptional events thereby preventing G(1)-S transition of renal cell cycle. Phosphorylation of IkappaBalpha was inhibited resulting in limited nuclear translocation of NF-kappaB. However, cyclin D1 expression was high probably due to transcriptional cooperation of AP-1. Cyclin D1/cyclin-dependent kinase 4 (cdk4)-cdk6 system-mediated phosphorylation of retinoblastoma protein was downregulated due to overexpression of p16 at 24 h after exposure to the lethal dose alone. Inhibition of S-phase stimulation was confirmed by proliferating cell nuclear antigen assay (PCNA). This inhibitory response was prevented if the lethal dose was administered 72 h after the low priming dose of DCVC due to promitogenic effect of the low dose. NF-kappaB-DNA binding is not limited if mice were pretreated with the priming dose. Cyclin D1/cdk4-cdk6 expression stimulated by the priming dose of DCVC was unaltered even after the lethal dose in the autoprotected group, explaining higher phosphorylated-pRB and S-phase stimulation found in this group. These results were corroborated with PCNA immunohistochemistry. These findings suggest that the priming dose relieves the block on compensatory tissue repair by upregulation of promitogenic mechanisms, normally blocked by the high dose when administered without the prior priming dose.

Evidence for an S-phase checkpoint regulating DNA replication after heat shock: a review.

Exposure of cells to heat inhibits a number of nuclear activities associated with semi-conservative replication of DNA including the incorporation of radiolabelled precursors into acid-insoluble DNA, the initiation of new replicons, the elongation of the DNA fibre at the replication fork, the synthesis and deposition of new histones into chromatin and the reorganization of nascent DNA into mature chromatin. These effects are likely to underlie the heat sensitivity of S-phase cells and may contribute to the radiosensitization observed in this phase of the cell cycle. While some of these effects may be explained as 'passive' consequences of heat-induced damage on chromatin structures experiments reviewed here point to the activation of a checkpoint as a contributing factor to the observed inhibition of DNA replication. Activation of a heat responsive S-phase checkpoint targets the activity of RPA via interaction with nucleolin. Nucleolin, a major nucleolar protein, is found normally sequestered in the nucleolus. Exposure of cells to heat causes a rapid translocation of nucleolin from the nucleolus into the nucleoplasm that enables RPA/nucleolin interaction. This interaction inhibits functions of RPA associated with the initiation of DNA replication and contributes to the immediate inhibition of DNA synthesis observed after heat shock. The results suggest that the nucleolus serves as a sequestration centre for the temporary inactivation of regulatory molecules, such as nucleolin, capable of regulating essential cellular functions after heat shock. It is speculated that this regulatory process is integrated in the network of responses that determine cell sensitivity to heat and that it may be involved in heat radiosensitization to killing as well.

Expression of flap endonuclease-1 during meiosis in a basidiomycete, Coprinus cinereus.

In the basidiomycete Coprinus cinereus (C. cinereus), which shows a highly synchronous meiotic cell cycle, the meiotic prophase I cells demonstrate flap endonuclease-1 activity. To investigate its role during meiosis, we isolated a C. cinereus cDNA homolog of flap endonuclease-1 (CcFEN-1), 1377bp in length with the open reading frame (ORF) encoding a predicted molecular mass of 51 kDa. At amino-acid residues Glu276-Pro345, a specific inserted sequence composed of 70 amino acids rich in polar forms was found to exist, without sequence identity to other eukaryotic FEN-1 or the polar amino acid rich sequences found in C. cinereus PCNA and C. cinereus DNA ligase IV, although the lengths and percentages of polar amino acids were similar. Northern hybridization analysis indicated CcFEN-1 to be expressed not only in the pre-meiotic S phase but also in meiotic prophase I. The roles of CcFEN-1 during meiosis are discussed.

The Yin Yang-1 (YY1) protein undergoes a DNA-replication-associated switch in localization from the cytoplasm to the nucleus at the onset of S phase.

The essential Yin Yang-1 gene (YY1) encodes a ubiquitous, conserved, multifunctional zinc-finger transcription factor in animals. The YY1 protein regulates initiation, activation, or repression of transcription from a variety of genes required for cell growth, development, differentiation, or tumor suppression, as well as from genes in some retroviruses and DNA viruses. Among the specific functions attributed to YY1 is a role in cell-cycle-specific upregulation of the replication-dependent histone genes. The YY1 protein binds to the histone alpha element, a regulatory sequence found in all replication-dependent histone genes. We therefore examined the abundance, DNA-binding activity and localization of the YY1 protein throughout the cell cycle in unperturbed, shake-off-synchronized Chinese hamster ovary and HeLa cells. We found that, whereas the DNA-binding activity of YY1 increased dramatically early in S phase, the YY1 mRNA and protein levels did not. YY1 changed subcellular distribution patterns during the cell cycle, from mainly cytoplasmic at G1 to mainly nuclear at early and middle S phase, then back to primarily cytoplasmic later in S phase. Nuclear accumulation of YY1 near the G1/S boundary coincided with both an increase in YY1 DNA-binding activity and the coordinate up-regulation of the replication-dependent histone genes. The DNA synthesis inhibitor aphidicolin caused a nearly complete loss of nuclear YY1, whereas addition of caffeine or 2-aminopurine to aphidicolin-treated cells restored both DNA synthesis and YY1 localization in the nucleus. These findings reveal a mechanism by which YY1 localization is coupled to DNA synthesis and responsive to cell-cycle signaling pathways. Taken together, our results provide insight into how YY1 might participate in the cell-cycle control over a variety of nuclear events required for cell division and proliferation.

Identification of HiNF-P, a key activator of cell cycle-controlled histone H4 genes at the onset of S phase.

At the G(1)/S phase cell cycle transition, multiple histone genes are expressed to ensure that newly synthesized DNA is immediately packaged as chromatin. Here we have purified and functionally characterized the critical transcription factor HiNF-P, which is required for E2F-independent activation of the histone H4 multigene family. Using chromatin immunoprecipitation analysis and ligation-mediated PCR-assisted genomic sequencing, we show that HiNF-P interacts with conserved H4 cell cycle regulatory sequences in vivo. Antisense inhibition of HiNF-P reduces endogenous histone H4 gene expression. Furthermore, we find that HiNF-P utilizes NPAT/p220, a substrate of the cyclin E/cyclin-dependent kinase 2 (CDK2) kinase complex, as a key coactivator to enhance histone H4 gene transcription. The biological role of HiNF-P is reflected by impeded cell cycle progression into S phase upon antisense-mediated reduction of HiNF-P levels. Our results establish that HiNF-P is the ultimate link in a linear signaling pathway that is initiated with the growth factor-dependent induction of cyclin E/CDK2 kinase activity at the restriction point and culminates in the activation of histone H4 genes through HiNF-P at the G(1)/S phase transition.

The ATM-related Tel1 protein of Saccharomyces cerevisiae controls a checkpoint response following phleomycin treatment.

MEC1 and TEL1 encode ATR- and ATM-related proteins in the budding yeast Saccharomyces cerevisiae, respectively. Phleomycin is an agent that catalyzes double-strand breaks in DNA. We show here that both Mec1 and Tel1 regulate the checkpoint response following phleomycin treatment. MEC1 is required for Rad53 phosphorylation and cell-cycle progression delay following phleomycin treatment in G1, S or G2/M phases. The tel1Delta mutation confers a defect in the checkpoint responses to phleomycin treatment in S phase. In addition, the tel1Delta mutation enhances the mec1 defect in activation of the phleomycin-induced checkpoint pathway in S phase. In contrast, the tel1Delta mutation confers only a minor defect in the checkpoint responses in G1 phase and no apparent defect in G2/M phase. Methyl methanesulfonate (MMS) treatment also activates checkpoints, inducing Rad53 phosphorylation in S phase. MMS-induced Rad53 phosphorylation is not detected in mec1Delta mutants during S phase, but occurs in tel1Delta mutants similar to wild-type cells. Finally, Xrs2 is phosphorylated after phleomycin treatment in a TEL1-dependent manner during S phase, whereas no significant Xrs2 phosphorylation is detected after MMS treatment. Together, our results support a model in which Tel1 contributes to checkpoint control in response to phleomycin-induced DNA damage in S phase.

Fish oil slows S phase progression and may cause upstream shift of DHFR replication origin ori-beta in CHO cells.

Fish oils (FOs) have been noted to reduce growth and proliferation of certain tumor cells, effects usually attributed to the content of polyunsaturated fatty acids of the n-3 family, which are thought to modulate cellular signaling pathways. We investigated the influence of FO on cell cycle kinetics of cultured Chinese hamster ovary cells. Exponentially growing cells were labeled with 5-bromo-2'-deoxyuridine (BrdU) and analyzed by flow cytometry after 5-day treatment with exogenous fat. Bivariate BrdU-DNA analysis indicated slower progression through S phase and thus longer S phase duration time in FO- but not corn oil-treated or control cells. We hypothesize that FO treatment might interfere with spatial/temporal organization of replication origins. Therefore, we mapped the well-characterized replication origin ori-beta downstream of the dihydrofolate reductase gene with the nascent strand length assay. Three DNA marker segments with known positions relative to this origin were amplified by PCR. By quantitatively assessing DNA length of the fragments in all fractions containing these markers, the location of ori-beta was established. In control or corn oil-treated cells, the location of ori-beta was consistent with previous studies. However, in FO-treated cells, DNA replication appears to start from a new site located farther upstream from ori-beta, suggesting a different replication initiation pattern. This study suggests novel mechanism(s) by which fats affect cell proliferation and DNA replication in mammalian cells.

AML1 stimulates G1 to S progression via its transactivation domain.

Inhibition of AML1-mediated transactivation potently slows G1 to S cell cycle progression. In Ba/F3 cells, activation of exogenous AML1 (RUNX1)-ER with 4-hydroxytamoxifen prevents inhibition of G1 progression mediated by CBFbeta-SMMHC, a CBF oncoprotein. We expressed three AML1-ER variants with CBFbeta-SMMHC in Ba/F3 cells. In these lines, CBFbeta-SMMHC expression is regulated by the zinc-responsive metallothionein promoter. Deletion of 72 AML1 C-terminal residues, which includes a transrepression domain, did not alter the activity of AML1-ER, whereas further deletion of 98 residues, removing the most potent AML1 transactivation domain (TAD), prevented rescue of cell cycle inhibition. Notably, the two variants which did not stimulate G1 exacerbated CBFbeta-SMMHC-mediated cell cycle arrest, suggesting that they dominantly inhibit AML1 activities. In addition, the two variants which stimulated G1 also induced apoptosis in 5-15% of the cells, an effect consistent with excessive G1 stimulation. These observations indicate that AML1 activates transcription of one or more genes critical for the G1 to S transition via its C-terminal transactivation domain. Inactivation of AML in acute leukemia is expected to slow proliferation unless additional genetic alterations co-exist which accelerate G1.

Prediction of node-negative breast cancer outcome by histologic grading and S-phase analysis by flow cytometry: an Eastern Cooperative Oncology Group Study (2192).

Histologic evaluation and reporting of invasive breast cancer has effectively used Nottingham combined histologic grade (NCHG). This approach to predict outcome in invasive breast cancer has not been tested in multicenter cooperative trials. Histologic slides from selected breast cancer cases entered on node-negative Eastern Cooperative Oncology Group trials were assigned grades. Two pathologists evaluated cases for NCHG defined from differentiation, mitotic index, and nuclear grade. The study population consisted of separate samples from low- and high-risk strata, where low risk was estrogen receptor positive with a tumor size of less than 3 cm and high risk was estrogen receptor negative or tumor size greater than or equal to 3 cm. The rate of agreement was generally good, with 80% of cases classified the same for mitotic count and 76% of the cases classified the same for combined grade. There were no cases disagreeing from the lowest to the highest of the three categories. The median follow-up is 11.6 years, but for analysis of survival, this was truncated at 5 years. Mitotic index and combined grade as assessed by both pathologists showed significant associations with survival. High combined histologic grade was predictive for response to cyclophosphamide/methotrexate/5-fluorouracil (CMF) with survival differences at 5 years of 30% in the treated high-grade patients over the untreated patients. Overall, it is clear that pathologists can have close agreement in assignment of combined histologic grades, with highly significant prediction in univariate and borderline significance in multivariate analysis in prognostication of time to recurrence as well as survival. Thus, stratification used in these trials was highly prognostic as hoped, leaving a role for histologic grading in these relatively large tumors, more powerful than S-phase analysis in this series. In the subgroups of high-risk patients randomized between CMF and observation, there was a suggestion that the high-combined-grade group was predictive of treatment efficacy. We conclude that a combined histologic grade with defined criteria may be reliably assigned by practiced pathologists using readily available criteria, and that the measure may be of use in prognostication and prediction of therapeutic responsiveness when done in a technically ideal fashion.