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Abstract Background The cellular retinol binding protein I gene (CRBP) is downregulated in a subset of human breast cancers and in MMTV-Myc induced mouse mammary tumors. Functional studies suggest that CRBP downregulation contributes to breast tumor progression. What is the mechanism underlying CRBP downregulation in cancer? Here we investigated the hypothesis that CRBP is epigenetically silenced through DNA hypermethylation in human and mouse breast cancer. Results Bisulfite sequencing of CRBP in a panel of 6 human breast cancer cell lines demonstrated that, as a rule, CRBP hypermethylation is closely and inversely related to CRBP expression and identified one exception to this rule. Treatment with 5-azacytidine, a DNA methyltransferase inhibitor, led to CRBP reexpression, supporting the hypothesis that CRBP hypermethylation is a proximal cause of CRBP silencing. In some cells CRBP reexpression was potentiated by co-treatment with retinoic acid, an inducer of CRBP, and trichostatin A, a histone deacetylase inhibitor. Southern blot analysis of a small panel of human breast cancer specimens identified one case characterized by extensive CRBP hypermethylation, in association with undetectable CRBP mRNA and protein. Bisulfite sequencing of CRBP in MMTV-Myc and MMTV-Neu/NT mammary tumor cell lines extended the rule of CRBP hypermethylation and silencing (both seen in MMTV-Myc but not MMTV-Neu/NT cells) from human to mouse breast cancer and suggested that CRBP hypermethylation is an oncogene-specific event. Conclusion CRBP hypermethylation appears to be an evolutionarily conserved and principal mechanism of CRBP silencing in breast cancer. Based on the analysis of transgenic mouse mammary tumor cells, we hypothesize that CRBP silencing in human breast cancer may be associated with a specific oncogenic signature.

Hemostasis describes the process of blood clotting homeostasis. Hemostasis reflects a balance of procoagulant and anticoagulant mechanisms that aim to prevent both bleeding and thrombosis. If hemostasis is disrupted, and bleeding or thrombosis occur, then laboratory testing may ensue to either diagnose the reason for bleeding or thrombosis, or to manage patients under therapy or treatment for bleeding or thrombosis. A wide range of tests of hemostasis are available to laboratories and to clinicians, from routine coagulation assays to specialized hemostasis assays and platelet function. In the current narrative review, we highlight some of the history of innovative diagnostic solutions, such as the integration of chemiluminescence and flow cytometry in the hemostasis diagnostic armamentarium, as well as providing a glimpse to the possible future of diagnostic hemostasis testing. Future directions include the potential for artificial intelligence in diagnostics, the development of more global test systems that can assess both primary and secondary hemostasis, and several innovations to enable the ongoing evolution of therapies to rebalance hemostasis and requiring precise monitoring. This review underscores the ongoing need for innovation to enhance the diagnostic landscape of hemostasis, ensuring better patient outcomes through more accurate and efficient diagnostic methods. [ABSTRACT FROM AUTHOR]

Background: Increasing evidence proves that RBP7 plays a significant role in breast cancer (BC). The present study was aimed to investigate the mechanism of RBP7. Methods: Western Blotting and qRT-PCR were performed for evaluating the expression levels. CCK8, colony forming, xenograft mouse model, wound healing and transwell assays were conducted to examine cell ability of proliferation, invasion and migration. Nile red staining and Oil red O staining were used for testing the lipid. Results: RBP7 was related to overall survival (OS) in patients with HR + BC. RBP7 protein was significantly decreased in HR + BC tissues and cells. RBP7 suppressed HR + BC cell proliferation in vitro and in vivo, and inhibited migration and invasion. RBP7 reduced fatty acid in HR + BC cells by inhibiting the AKT/SREBP1 pathway. Conclusions: RBP7 may function as a tumor suppressor in HR + BC by inhibiting the AKT/SREBP1 pathway and reducing fatty acid. [ABSTRACT FROM AUTHOR]

There is accumulating evidence that vitamin A (VA) deficiency contributes to the pathogenesis and progression of Alzheimer's disease (AD). All-trans retinoic acid (ATRA), a metabolite of VA in the brain, serves distinct roles in the human hippocampus. Agonists of retinoic acid receptors (RAR), including ATRA, promote activation of the non-amyloidogenic pathway by enhancing expression of α-secretases, providing a mechanistic basis for delaying/preventing amyloid beta (Aβ) toxicity. However, whether ATRA is actually deficient in the hippocampi of patients with AD is not clear. Here, using a publicly available human transcriptomic dataset, we evaluated the extent to which ATRA-sensitive genes are dysregulated in hippocampal tissue from post-mortem AD brains, relative to age-matched controls. Consistent with ATRA deficiency, we found significant dysregulation of many ATRA-sensitive genes and significant upregulation of RAR co-repressors, supporting the idea of transcriptional repression of ATRA-mediated signaling. Consistent with oxidative stress and neuroinflammation, Nrf2 and NfkB transcripts were upregulated, respectively. Interestingly, transcriptional targets of Nrf2 were not upregulated, accompanied by upregulation of several histone deacetylases. Overall, our investigation of ATRA-sensitive genes in the human hippocampus bolsters the scientific premise of ATRA depletion in AD and that epigenetic factors should be considered and addressed as part of VA supplementation. [ABSTRACT FROM AUTHOR]

The vascular endothelium has several important functions, including hemostasis. The homeostasis of hemostasis is based on a fine balance between procoagulant and anticoagulant proteins and between fibrinolytic and antifibrinolytic ones. Coagulopathies are characterized by a mutation-induced alteration of the function of certain coagulation factors or by a disturbed balance between the mechanisms responsible for regulating coagulation. Homeostatic therapies consist in replacement and nonreplacement treatments or in the administration of antifibrinolytic agents. Rebalancing products reestablish hemostasis by inhibiting natural anticoagulant pathways. These agents include monoclonal antibodies, such as concizumab and marstacimab, which target the tissue factor pathway inhibitor; interfering RNA therapies, such as fitusiran, which targets antithrombin III; and protease inhibitors, such as serpinPC, which targets active protein C. In cases of thrombophilia (deficiency of protein C, protein S, or factor V Leiden), treatment may consist in direct oral anticoagulants, replacement therapy (plasma or recombinant ADAMTS13) in cases of a congenital deficiency of ADAMTS13, or immunomodulators (prednisone) if the thrombophilia is autoimmune. Monoclonal-antibody-based anti-vWF immunotherapy (caplacizumab) is used in the context of severe thrombophilia, regardless of the cause of the disorder. In cases of disseminated intravascular coagulation, the treatment of choice consists in administration of antifibrinolytics, all-trans-retinoic acid, and recombinant soluble human thrombomodulin. [ABSTRACT FROM AUTHOR]

Nephropathic cystinosis (NC) is a rare autosomal recessive disease, which causes cysteine-crystals accumulation with progression to end-stage renal disease (ESRD). Von willebrand disease (VWD) type III is a rare subtype of von willebrand factor (VWF) abnormality, which is characterized by severe reduction of VWF and factor VIII activity. A 16-year-old patient with NC and VWD type III presented with uremic symptoms due to ESRD. Dialysis access was inserted and followed by hemodialysis (HD) for 4 months with a proper infusion of blood products. While renal transplant remains the treatment of choice of NC and superior to chronic HD, bleeding complications were a major concern in this case with coexisting VWD type III. However, with the meticulous implementation of the Hematology team's daily recommendations, renal transplantation was successfully performed. This is the first case that mentions a new association between two inherited rare disorders, NC and VWD type III, and this entity has not been reported before. Moreover, successful kidney transplantation in our patient supports the possibility of these procedures in hereditary clotting disorders. [ABSTRACT FROM AUTHOR]

Purpose: In this study the effectiveness of encapsulating of 5-azacytidine into the lipid nanoparticles was investigated and in vitro effect of encapsulated 5-azacytidine studied on MCF-7 cell lines Methods: 5-azacytidine-loaded solid lipid nanoparticles were produced by double emulsification (w/o/w) method by using stearic acid as lipid matrix, soy lecithin and poloxamer 407 as surfactant and co-surfactant respectively. Particle size, zeta potential, surface morphology, entrapment efficiency and kinetic of drug release were studied. In vitro effect of 5-azacytidine on MCF-7 cell line studied by MTT assay, DAPI staining, Rhodamine B relative uptake, and also Real time RT-PCR was performed for studying difference effect of free and encapsulated drug on expression of RARß2 gene. Results: The formulation F5 with 55.84±0.46 % of entrapment efficiency shows zero order kinetic of drug release and selected for in vitro studies; the cytotoxicity of free drug and encapsulated drug in 48 h of incubation have significant difference. DAPI staining shows morphology of apoptotic nucleus in both free and encapsulated drug, Rhodamine B labeled SLNs show time dependency and accumulation of SLNs in cytoplasm. Real time qRT-PCR doesn't show any significant difference (p>0.05) in expression of RARß2 gene in both cells treated with free or encapsulated drug. Conclusion: The results of the present study indicated that the entrapment of 5-azacytidine into SLNs enhanced its cytotoxicity performance and may pave a way for the future design of a desired dosage form for 5-azacytidine. [ABSTRACT FROM AUTHOR]

Vitamin A is an essential diet-derived nutrient that has biological activity affected through an active metabolite, all-trans retinoic acid (atRA). Retinol-binding protein type 1 (RBP1) is an intracellular chaperone that binds retinol and retinal with high affinity, protects retinoids from non-specific oxidation, and delivers retinoids to specific enzymes to facilitate biosynthesis of RA. RBP1 expression is reduced in many of the most prevalent cancers, including breast cancer. Here, we sought to understand the relationship between RBP1 expression and atRA biosynthesis in mammary epithelial cells, as well as RBP1 expression and atRA levels in human mammary tissue. We additionally aimed to investigate the impact of RBP1 expression and atRA on the microenvironment as well as the potential for therapeutic restoration of RBP1 expression and endogenous atRA production. Using human mammary ductal carcinoma samples and a series of mammary epithelial cell lines representing different stages of tumorigenesis, we investigated the relationship between RBP1 expression as determined by QPCR and atRA via direct liquid chromatography-multistage-tandem mass spectrometry-based quantification. The functional effect of RBP1 expression and atRA in epithelial cells was investigated via the expression of direct atRA targets using QPCR, proliferation using Ki-67 staining, and collagen deposition via picrosirius red staining. We also investigated the atRA content of stromal cells co-cultured with normal and tumorigenic epithelial cells. Results show that RBP1 and atRA are reduced in mammary tumor tissue and tumorigenic epithelial cell lines. Knock down of RBP1 expression using shRNA or overexpression of RBP1 supported a direct relationship between RBP1 expression with atRA. Increases in cellular atRA were able to activate atRA direct targets, inhibit proliferation and inhibit collagen deposition in epithelial cell lines. Conditions encountered in tumor microenvironments, including low glucose and hypoxia, were able to reduce RBP1 expression and atRA. Treatment with either RARα agonist AM580 or demethylating agent Decitabine were able to increase RBP1 expression and atRA. Cellular content of neighboring fibroblasts correlated with the RA producing capacity of epithelial cells in co-culture. This work establishes a direct relationship between RBP1 expression and atRA, which is maintained when RBP1 expression is restored therapeutically. The results demonstrate diseases with reduced RBP1 could potentially benefit from therapeutics that restore RBP1 expression and endogenous atRA. [ABSTRACT FROM AUTHOR]

Introduction: Current hormonal adjuvant therapies for breast cancer including tamoxifen treatment and estrogen depletion are overall tumoristatic and are severely limited by the frequent recurrence of the tumors. Regardless of the resistance mechanism, development and progression of the resistant tumors requires the persistence of a basal level of cycling cells during the treatment for which the underlying causes are unclear. Methods: In estrogen-sensitive breast cancer cells the effects of hormone depletion and treatment with estrogen, tamoxifen, all-trans retinoic acid (ATRA), fulvestrant, estrogen receptor α (ER) siRNA or retinoic acid receptor α (RARα) siRNA were studied by examining cell growth and cycling, apoptosis, various mRNA and protein expression levels, mRNA profiles and known chromatin associations of RAR. RARα subtype expression was also examined in breast cancer cell lines and tumors by competitive PCR. Results: Basal proliferation persisted in estrogen-sensitive breast cancer cells grown in hormone depleted conditioned media without or with 4-hydroxytamoxifen (OH-Tam). Downregulating ER using either siRNA or fulvestrant inhibited basal proliferation by promoting cell cycle arrest, without enrichment for ErbB2/3+ overexpressing cells. The basal expression of RARα1, the only RARα isoform that was expressed in breast cancer cell lines and in most breast tumors, was supported by apo-ER but was unaffected by OH-Tam; RAR-β and -γ were not regulated by apo-ER. Depleting basal RARα1 reproduced the antiproliferative effect of depleting ER whereas its restoration in the ER depleted cells partially rescued the basal cycling. The overlapping tamoxifen-insensitive gene regulation by apo-ER and apo-RARα1 comprised activation of mainly genes promoting cell cycle and mitosis and suppression of genes involved in growth inhibition; these target genes were generally insensitive to ATRA but were enriched in RAR binding sites in associated chromatin regions. Conclusions: In hormone-sensitive breast cancer, ER can support a basal fraction of S-phase cells (i) without obvious association with ErbB2/3 expression, (ii) by mechanisms unaffected by hormone depletion or OH-Tam and (iii) through maintenance of the basal expression of apo-RARα1 to regulate a set of ATRA-insensitive genes. Since isoform 1 of RARα is genetically redundant, its targeted inactivation or downregulation should be further investigated as a potential means of enhancing hormonal adjuvant therapy. [ABSTRACT FROM AUTHOR]

Most neuroblastoma patients over 18 months of age at diagnosis present disseminated disease. The presence of neuroblastoma cells in bone marrow can be used to evaluate the response to treatment. It is possible that alterations in certain tumour cells might confer a selective advantage over tumour dissemination process, and probably be helpful in the clonal selection of tumour-specific cells that could originate metastasis. We performed real-time quantitative PCR to identify the presence of disseminated tumour cells in bone marrow samples, and we used MSP to analyse the methylation profile of 20 genes putatively implied in dissemination. We described epigenetic alterations in the methylated status of certain genes in disseminated tumour cells from bone marrow. Those cases with high rate of hypermethylation showed an increased probability of relapse during or after treatment. We found significantly poor prognosis in event-free survival in cases with hypermethylation of TMS1, MGMT and RARβ2 genes. We could not confirm the presence of a specific methylation profile in disseminated neuroblastoma tumour cells, but a high accumulation of epigenetic events in those cells is associated with a high risk of relapse, independently of MYCN amplification. [ABSTRACT FROM AUTHOR]

To determine the hypermethylation status of the promoter regions of tumour suppressor genes in breast tissues from healthy women and identify the determinants of these epigenetic changes. Questionnaires and breast tissues were collected from healthy women without a history of cancer and undergoing reduction mammoplasty ( N= 141). Methylation for p16INK4, BRCA1, ERα and RAR-β promoter regions from breast tissues were determined by methylation specific PCR. Associations were examined with chi-square and Fisher’s exact test as well as logistic regression. All statistical tests were two-sided. p16INK4, BRCA1, ERα and RAR-β hypermethylation were identified in 31%, 17%, 9% and 0% of the women, respectively. Women with BRCA1 hypermethylation had an eight-fold increase in the risk of ERα hypermethylation ( P= 0.007). p16INK4 hypermethylation was present in 28% of African-Americans, but 65% in European-Americans ( P= 0.02). There was an increased likelihood of p16INK4 or BRCA1 hypermethylation for women with family history of cancer (OR 2.3; 95%CI: 1.05–4.85 and OR 5.0; 95%CI: 1.55–15.81, respectively). ERα hypermethylation was associated with family history of breast cancer (OR 6.6; 95%CI: 1.58–27.71). After stratification by race, p16INK4 in European-Americans and BRCA1 hypermethylation in African-Americans were associated with family history of cancer (OR 3.8; 95%CI: 1.21–12.03 and OR 6.5; 95%CI: 1.33–31.32, respectively). Gene promoter hypermethylation was commonly found in healthy breast tissues from women without cancer, indicating that these events are frequent and early lesions. Race and family history of cancer increase the likelihood of these early events. [ABSTRACT FROM AUTHOR]

Background: We recently have shown that Charged multivesicular protein/Chromatin modifying protein1A (Chmp1A) functions as a tumor suppressor in human pancreatic tumor cells. Pancreatic cancer has the worst prognosis of all cancers with a dismal 5-year survival rate. Preclinical studies using ATRA for treating human pancreatic cancer suggest this compound might be useful for treatment of pancreatic cancer patients. However, the molecular mechanism by which ATRA inhibits growth of pancreatic cancer cells is not clear. The objective of our study was to investigate whether Chmp1A is involved in ATRA-mediated growth inhibition of human pancreatic tumor cells. Results: We performed microarray studies using HEK 293T cells and discovered that Chmp1A positively regulated Cellular retinol-binding protein 1 (CRBP-1). CRBP-1 is a key regulator of All-trans retinoic acid (ATRA) through ATRA metabolism and nuclear localization. Since our microarray data indicates a potential involvement of Chmp1A in ATRA signaling, we tested this hypothesis by treating pancreatic tumor cells with ATRA in vitro. In the ATRA-responsive cell lines, ATRA significantly increased the protein expression of Chmp1A, CRBP-1, P53 and phospho-P53 at serine 15 and 37 position. We found that knockdown of Chmp1A via shRNA abolished the ATRA-mediated growth inhibition of PanC-1 cells. Also, Chmp1A silencing diminished the increase of Chmp1A, P53 and phospho-P53 protein expression induced by ATRA. In the ATRA non-responsive cells, ATRA did not have any effect on the protein level of Chmp1A and P53. Chmp1A over-expression, however, induced growth inhibition of ATRA non-responsive cells, which was accompanied by an increase of Chmp1A, P53 and phospho-P53. Interestingly, in ATRA responsive cells Chmp1A is localized to the nucleus, which became robust upon ATRA treatment. In the ATRA-non-responsive cells, Chmp1A was mainly translocated to the plasma membrane upon ATRA treatment. Conclusion: Collectively our data provides evidence that Chmp1A mediates the growth inhibitory activity of ATRA in human pancreatic cancer cells via regulation of CRBP-1. Our results also suggest that nuclear localization of Chmp1A is important in mediating ATRA signaling. [ABSTRACT FROM AUTHOR]

Antitumour effects of retinoids are attributed to their influence on cell proliferation, differentiation, apoptosis and angiogenesis. In our effort to develop useful agents for breast cancer therapy, we evaluated the effects of four representative retinoic acid metabolism blocking agents (RAMBAs, VN/14-1, VN/50-1, VN/66-1 and VN/69-1) on growth inhibition of oestrogen receptor positive (ER +ve, MCF-7 and T-47D) and oestrogen receptor negative (ER −ve, MDA-MB-231) human breast cancer cells. Additionally, we investigated the biological effects/molecular mechanism(s) underlying their growth inhibitory properties as well as their antitumour efficacies against MCF-7 and MCF-7Ca tumour xenografts in nude mice. We also assessed the effect of combining VN/14-1 and all-trans-retinoic acid (ATRA) on MCF-7 tumuor xenografts. The ER +ve cell lines were more sensitive (IC50 values between 3.0 and 609 nM) to the RAMBAs than the ER −ve MDA-MB-231 cell line (IC50=5.6–24.0 μM). Retinoic acid metabolism blocking agents induced cell differentiation as determined by increased expression of cytokeratin 8/18 and oestrogen receptor-α (ER-α). Similar to ATRA, they also induced apoptosis via activation of caspase 9. Cell cycle analysis indicated that RAMBAs arrested cells in the G1 and G2/M phases and caused significant downregulation (>80%) of cyclin D1 protein. In vivo, the growth of MCF-7 mammary tumours was dose-dependently and significantly inhibited (92.6%, P<0.0005) by VN/14-1. The combination of VN/14-1 and ATRA also inhibited MCF-7 breast tumour growth in vivo (up to 120%) as compared with single agents (P<0.025). VN/14-1 was also very effective in preventing the formation of MCF-7Ca tumours and it significantly inhibited the growth of established MCF-7Ca tumours, being as effective as the clinically used aromatase inhibitors, anastrozole and letrozole. Decrease in cyclin D1 and upregulation of cytokeratins, Bad and Bax with VN/14-1 may be responsible for the efficacy of this compound in inhibiting breast cancer cell growth in vitro and in vivo. Our results suggest that our RAMBAs, especially VN/14-1 may be useful novel therapy for breast cancer.British Journal of Cancer (2007) 96, 1204–1215. doi:10.1038/sj.bjc.6603705 www.bjcancer.com Published online 27 March 2007 [ABSTRACT FROM AUTHOR]

Background: Epidemiological studies on risk factors for colorectal cancer (CRC) have mainly focused on diet, and being overweight is now recognized to contribute significantly to CRC risk. Overweight and obesity are defined as an excess of adipose tissue mass and are associated with disorders in lipid metabolism. Peroxisome proliferator-activated receptors (PPARs) and retinoid-activated receptors (RARs and RXRs) are important modulators of lipid metabolism and cellular homeostasis. Alterations in expression and activity of these ligand-activated transcription factors might be involved in obesity-associated diseases, which include CRC. Cyclooxygenase-2 (COX-2) also plays a critical role in lipid metabolism and alterations in COX-2 expression have already been associated with unfavourable clinical outcomes in epithelial tumors. The objective of this study is to examine the hypothesis questioning the relationship between alterations in the expression of nuclear receptors and COX-2 and the weight status among male subjects with CRC. Method: The mRNA expression of the different nuclear receptor subtypes and of COX-2 was measured in 20 resected samples of CRC and paired non-tumor tissues. The association between expression patterns and weight status defined as a body mass index (BMI) was statistically analyzed. Results: No changes were observed in PPARγ mRNA expression while the expression of PPARδ, retinoid-activated receptors and COX-2 were significantly increased in cancer tissues compared to normal colon mucosa (P ≤ 0.001). The weight status appeared to be an independent factor, although we detected an increased level of COX-2 expression in the normal mucosa from overweight patients (BMI ≥ 25) compared to subjects with healthy BMI (P = 0.002). Conclusion: Our findings show that alterations in the pattern of nuclear receptor expression observed in CRC do not appear to be correlated with patient weight status. However, the analysis of COX-2 expression in normal colon mucosa from subjects with a high BMI suggests that COX-2 deregulation might be driven by excess weight during the colon carcinogenesis process. [ABSTRACT FROM AUTHOR]

Background: Retinoic acid suppresses cell growth and promotes cell differentiation, and pharmacological retinoic acid receptor (RAR) activation is anti-tumorigenic. This begs the question of whether chronic physiological RAR activation by endogenous retinoids is likewise antitumorigenic. Results: To address this question, we generated transgenic mice in which expression of a ligand binding defective dominant negative RARá (RARáG303E) was under the control of the mouse mammary tumor virus (MMTV) promoter. The transgene was expressed in the lymphoid compartment and in the mammary epithelium. Observation of aging mice revealed that transgenic mice, unlike their wild type littermates, developed B cell lymphomas at high penetrance, with a median latency of 40 weeks. MMTV-RARáG303E lymphomas were high grade Pax-5+, surface H+L Ig negative, CD69+ and BCL6- and cytologically and phenotypically resembled human adult high grade (Burkitt's or lymphoblastic) lymphomas. We postulated that mammary tumors might arise after a long latency period as seen in other transgenic models of breast cancer. We tested this idea by transplanting transgenic epithelium into the cleared fat pads of wild type hosts, thus bypassing lymphomagenesis. At 17 months post-transplantation, a metastatic mammary adenocarcinoma developed in one of four transplanted glands whereas no tumors developed in sixteen of sixteen endogenous glands with wild type epithelium. Conclusion: These findings suggest that physiological RAR activity may normally suppress B lymphocyte and mammary epithelial cell growth and that global RAR inactivation is sufficient to initiate a stochastic process of tumor development requiring multiple transforming events. Our work makes available to the research community a new animal resource that should prove useful as an experimental model of aggressive sporadic lymphoma in immunologically uncompromised hosts. We anticipate that it may also prove useful as a model of breast cancer. [ABSTRACT FROM AUTHOR]

The retinoic acid receptor alpha (RARA) gene is disrupted by PML/RARA fusion in acute promyelocytic leukemia (APL). The P2 promoter of RARA, controlling the RARalpha2 isoform, contains an RA-responsive element and may be targeted in APL. To test whether aberrant methylation of P2 was involved, 47 APL at diagnosis, 16 APL at first relapse, 50 acute myeloid leukemia (AML) and 22 acute lymphoblastic leukemia (ALL) were tested by methylation-specific polymerase chain reaction. RARA P2 methylation was highly associated with APL (APL: 25/63 vs AML/ALL: 2/75, P<0.0001). P2 methylation occurred at similar frequencies in APL at diagnosis and relapse, suggesting it was an initiating leukemogenic event. In the APL line NB4, RARalpha2 was not expressed, with the untranslocated RARA shown to be P2 methylated. 5-Azacytadine treatment of NB4 led to progressive P2 demethylation and re-expression of RARalpha2, confirming that RARA methylation collaborated with PML/RARA in totally suppressing RARalpha. In APL, RARA P2 methylation was unrelated to gender, age, presenting leukocyte counts and additional cytogenetic aberrations. For APL patients receiving all-trans retinoic acid for induction, P2 methylation did not affect the complete remission rates and survivals. RARA is the first myeloid-specific transcription factor shown to be dysregulated by both translocation and aberrant methylation. [ABSTRACT FROM AUTHOR]

Background: Retinoic acid receptor (RAR) activation induces cell differentiation and may antagonize cancer progression. Cellular retinol-binding protein I (CRBP-I) functions in retinol storage and its expression is lower in human cancers than in normal cells. We hypothesized that retinol storage might be linked to RAR activation and thus that lowered CRBP-I function might impair RAR activity and cell differentiation.Methods: Sarcoma virus 40-immortalized human mammary epithelial cells (MTSV1-7) devoid of CRBP-I were transfected with wild-type CRBP-I or CRBP-I point mutants with low RA binding affinity. The subcellular localization of CRBP-I was investigated in these cells and in wild-type or CRBP-I null mouse mammary epithelial cells (MECs), using indirect immunofluorescence and sucrose gradient fractionation. RAR activity was assessed using reporter gene assays. Acinar differentiation and in vivo tumor growth were assessed in reconstituted basement membrane and athymic mice, respectively.Results: In cells expressing wild-type CRBP-I but not the CRBP-I mutants, CRBP-I was found mainly in lipid droplets, the retinol storage organelle, and this localization was associated with promotion of retinol storage by wild-type CRBP-I only. RAR activity was higher and acinar differentiation was observed in cells expressing wild-type but not mutant CRBP-I. RAR antagonist treatment blocked and chronic RA treatment mimicked, the CRBP-I induction of cell differentiation. Finally, CRBP-I suppressed tumorigenicity in athymic mice.Conclusions: Physiologic RAR activation is dependent on CRBP-I-mediated retinol storage, and CRBP-I downregulation chronically compromises RAR activity, leading to loss of cell differentiation and tumor progression. [ABSTRACT FROM AUTHOR]

In mammalian cells, the SWI-SNF chromatin-remodeling complex is a regulator of cell proliferation, and overexpression of the catalytic subunit Brm interferes with cell cycle progression. Here, we show that treatment with histone deacetylase (HDAC) inhibitors reduces the inhibitory effect of Brm on the growth of mouse fibroblasts. This observation led to the identification of two carboxy-terminal acetylation sites in the Brm protein. Mutation of these sites into non-acetylatable sequences increased both the growth-inhibitory and the transcriptional activities of Brm. We also show that culture in the presence of HDAC inhibitors facilitates the isolation of clones overexpressing Brm. Removal of the HDAC inhibitors from the growth medium of these clones leads to downregulation of cyclin D1. This downregulation is absent in cell transformed by oncogenic ras. [ABSTRACT FROM AUTHOR]

Discusses the role of DNA methylation in breast carcinogenesis. Details on DNA methylation and the establishment of six novel capabilities towards breast carcinogenesis; Information on DNA methylation, memory and breast carcinogenesis; Prevention of breast cancer.

Investigates the effect of histone deacetylases (HDAC) inhibitors on different models of angiogenesis. Information on angiogenesis; Function of HDAC; Types of HDAC inhibitors; Factors that contribute to the blocking of HDAC activity.

We showed earlier that cellular retinol-binding protein (CRBP) expression is downregulated in a subset of human breast cancers. We have now investigated the outcome of ectopic CRBP expression in MTSV1-7 cells, a SV40 T antigen-transformed human breast epithelial cell line devoid of endogenous CRBP expression. We found that: (i) CRBP did not inhibit adherent cell growth but suppressed foci formation in post-confluent cultures and colony formation in soft agar; (ii) this effect was due to CRBP inhibition of cell survival, as demonstrated by viability and TUNEL assays of cells in soft-agar or plated on polyHEMA-coated dishes; (iii) CRBP inhibited protein kinase B/Akt activation in cells in suspension but not in adherent cells and the CRBP suppression of anchorage-independent growth was mimicked by cell treatment with the phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002; (iv) CRBP enhanced retinyl ester formation and storage but did not regulate retinoic acid synthesis or retinoic acid receptor activity. Ectopic CRBP-mediated inhibition of anchorage-independent cell survival and colony formation in the absence of significantly altered responses to either retinol or retinoic acid was also documented in T47D human breast cancer cells. In conclusion, the data suggest two novel and linked CRBP functions in mammary epithelial cells: inhibition of the PI3K/Akt survival pathway and suppression of anchorage-independent growth. Oncogene (2001) 20, 7413–7419. [ABSTRACT FROM AUTHOR]

DNA methylation is not just for basic scientists any more. There is a growing awareness in the medical field that having the correct pattern of genomic methylation is essential for healthy cells and organs. If methylation patterns are not properly established or maintained, disorders as diverse as mental retardation, immune deficiency, and sporadic or inherited cancers may follow. Through inappropriate silencing of growth regulating genes and simultaneous destabilisation of whole chromosomes, methylation defects help create a chaotic state from which cancer cells evolve. Methylation defects are present in cells before the onset of obvious malignancy and therefore cannot be explained simply as a consequence of a deregulated cancer cell. Researchers are now able to detect with exquisite sensitivity the cells harbouring methylation defects, sometimes months or years before the time when cancer is clinically detectable. Furthermore, aberrant methylation of specific genes has been directly linked with the tumour response to chemotherapy and patient survival. Advances in our ability to observe the methylation status of the entire cancer cell genome have led us to the unmistakable conclusion that methylation abnormalities are far more prevalent than expected. This methylomics approach permits the integration of an ever growing repertoire of methylation defects with the genetic alterations catalogued from tumours over the past two decades. Here we discuss the current knowledge of DNA methylation in normal cells and disease states, and how this relates directly to our current understanding of the mechanisms by which tumours arise. [ABSTRACT FROM AUTHOR]

A growing body of evidence supports the hypothesis that the retinoic acid receptor β2 (RAR-β2) gene is a tumor suppressor gene which induces apoptosis and that the chemopreventive and therapeutic effects of retinoids are due to induction of RAR-β2. During breast cancer progression, RAR-β2 is reduced or even lost. It is known from studies of other tumor-suppressor genes that methylation of the 5′-region is the cause of loss of expression. Several groups demonstrated that this is also true for the RAR-β2 in breast cancer by treating breast cancer cell lines with a demethylating agent and examining expression of the RAR-β2 gene in response to a challenge with retinoic acid. Studies using sodium bisulfite genomic sequencing as well as methylation specific PCR showed that a number of breast cancer cell lines as well as breast cancer tissue showed signs of methylation. The RAR-β2 gene was unmethylated in non-neoplastic breast tissue as well as in other normal tissues. A combination of retinoic acid with demethylating agents as well as with histone deacetylase inhibitors acts synergistically to inhibit growth. This review presents data that suggest that treatment of cancer patients with demethylating agents followed by retinoic acid may offer a new therapeutic modality. Both the time of commencement of chemoprevention and the choice of substances that are able either to prevent de novo methylation or to reverse methylation-caused gene silencing may be important considerations. [ABSTRACT FROM AUTHOR]

Presents an author index for the December 27, 2000 issue of the book 'Oncogene.'

Although the use of oral administration of pharmacological all-trans retinoic acid (ATRA) concentration in acute promyelocytic leukaemia (APL) patients was approved for over 20 years and used as standard therapy still to date, the same use in solid cancers is still controversial. In the present review the literature about the top five lethal solid cancers (lung, stomach, liver, breast, and colon cancer), as defined by The Global Cancer Observatory of World Health Organization, and retinoic acids (ATRA, 9-cis retinoic acid, and 13-cis retinoic acid, RA) was compared. The action of retinoic acids in inhibiting the cell proliferation was found in several cell pathways and compartments: from membrane and cytoplasmic signaling, to metabolic enzymes, to gene expression. However, in parallel in the most aggressive phenotypes several escape routes have evolved conferring retinoic acids-resistance. The comparison between different solid cancer types pointed out that for some cancer types several information are still lacking. Moreover, even though some pathways and escape routes are the same between the cancer types, sometimes they can differently respond to retinoic acid therapy, so that generalization cannot be made. Further studies on molecular pathways are needed to perform combinatorial trials that allow overcoming retinoic acids resistance. [ABSTRACT FROM AUTHOR]

An epigenetic component, especially aberrant DNA methylation pattern, has been shown to be frequently involved in sporadic breast cancer development. A growing body of literature demonstrates that combination of agents, i.e. nucleoside analogues with dietary phytochemicals, may provide enhanced therapeutic effects in epigenetic reprogramming of cancer cells. Clofarabine (2-chloro-2′-fluoro-2′-deoxyarabinosyladenine, ClF), a second-generation 2′-deoxyadenosine analogue, has numerous anti-cancer effects, including potential capacity to regulate epigenetic processes. Our present study is the first to investigate the combinatorial effects of ClF (used at IC50 concentration) with epigallocatechin-3-gallate (EGCG, tea catechin) or genistein (soy phytoestrogen), at physiological concentrations, on breast cancer cell growth, apoptosis, and epigenetic regulation of retinoic acid receptor beta (RARB) transcriptional activity. In MCF7 and MDA-MB-231 cells, RARB promoter methylation and expression of RARB, modifiers of DNA methylation reaction (DNMT1, CDKN1A, TP53), and potential regulator of RARB transcription, PTEN, were estimated using methylation-sensitive restriction analysis (MSRA) and quantitative real-time polymerase chain reaction (qPCR), respectively. The combinatorial exposures synergistically or additively inhibited the growth and induced apoptosis of breast cancer cells, followed by RARB hypomethylation with concomitant multiple increase in RARB, PTEN, and CDKN1A transcript levels. Taken together, our results demonstrate the ability of ClF-based combinations with polyphenols to promote cancer cell death and reactivate DNA methylation-silenced tumor suppressor genes in breast cancer cells with different invasive potential. [ABSTRACT FROM AUTHOR]