Higher order genomic organization and epigenetic control maintain cellular identity and prevent breast cancer.

Cells establish and sustain structural and functional integrity of the genome to support cellular identity and prevent malignant transformation. In this review, we present a strategic overview of epigenetic regulatory mechanisms including histone modifications and higher order chromatin organization (HCO) that are perturbed in breast cancer onset and progression. Implications for dysfunctions that occur in hormone regulation, cell cycle control, and mitotic bookmarking in breast cancer are considered, with an emphasis on epithelial-to-mesenchymal transition and cancer stem cell activities. The architectural organization of regulatory machinery is addressed within the contexts of translating cancer-compromised genomic organization to advances in breast cancer risk assessment, diagnosis, prognosis, and identification of novel therapeutic targets with high specificity and minimal off target effects.

Gut-derived serotonin contributes to bone deficits in colitis.

Osteoporosis and bone fractures occur at higher frequency in patients with inflammatory bowel disease (IBD), and decreased bone mass is observed in animal models of colitis. Another consistent feature of colitis is increased serotonin (5-HT) availability in the intestinal mucosa. Since gut-derived 5-HT can decrease bone mass, via activation of 5-HT1B receptors on pre-osteoblasts, we tested the hypothesis that 5-HT contributes to bone loss in colitis. Colitis was chronically induced in mice by adding dextran sodium sulfate (DSS) to their drinking water for 21 days. At day 21, circulating 5-HT levels were elevated in DSS-inflamed mice. Micro-computed tomography of femurs showed a decrease in trabecular bone volume fraction, formation, and surface area, due largely to decreased trabecular numbers in DSS-treated mice. The colitis-induced loss of trabecular bone was significantly suppressed in mice treated with the 5-HT synthesis inhibitor, p-chloro-DL-phenylalanine (PCPA; 300 mg/kg/day IP daily), and in mice treated with the 5-HT1B receptor antagonist GR55562 (1 mg/Kg/day SC daily). The 5-HT reuptake transporter (SERT) is critical for moving 5-HT from the interstitial space into enterocytes and from serum into platelets. Mice lacking SERT exhibited significant deficits in trabecular bone mass that are similar to those observed in DSS-inflamed mice, and these deficits were not extensively worsened by DSS-induced colitis in the SERT-/- mice. Taken together, findings from both the DSS and SERT-/- mouse models support a contributing role for 5-HT as a significant factor in bone loss induced by colitis.

Bicalutamide-induced hypoxia potentiates RUNX2-mediated Bcl-2 expression resulting in apoptosis resistance.

BACKGROUND: We have previously shown that hypoxia selects for more invasive, apoptosis-resistant LNCaP prostate cancer cells, with upregulation of the osteogenic transcription factor RUNX2 and the anti-apoptotic factor Bcl-2 detected in the hypoxia-selected cells. Following this observation, we questioned through what biological mechanism this occurs. METHODS: We examined the effect of hypoxia on RUNX2 expression and the role of RUNX2 in the regulation of Bcl-2 and apoptosis resistance in prostate cancer. RESULTS: Hypoxia increased RUNX2 expression in vitro, and bicalutamide-treated LNCaP tumours in mice (previously shown to have increased tumour hypoxia) exhibited increased RUNX2 expression. In addition, RUNX2-overexpressing LNCaP cells showed increased cell viability, following bicalutamide and docetaxel treatment, which was inhibited by RUNX2 siRNA; a range of assays demonstrated that this was due to resistance to apoptosis. RUNX2 expression was associated with increased Bcl-2 levels, and regulation of Bcl-2 by RUNX2 was confirmed through chromatin immunoprecipitation (ChIP) binding and reporter assays. Moreover, a Q-PCR array identified other apoptosis-associated genes upregulated in the RUNX2-overexpressing LNCaP cells. CONCLUSION: This study establishes a contributing mechanism for progression of prostate cancer cells to a more apoptosis-resistant and thus malignant phenotype, whereby increased expression of RUNX2 modulates the expression of apoptosis-associated factors, specifically Bcl-2.

Targeted inhibition of mitochondrial Hsp90 suppresses localised and metastatic prostate cancer growth in a genetic mouse model of disease.

BACKGROUND: The molecular chaperone heat shock protein-90 (Hsp90) is a promising cancer drug target, but current Hsp90-based therapy has so far shown limited activity in the clinic. METHODS: We tested the efficacy of a novel mitochondrial-targeted, small-molecule Hsp90 inhibitor, Gamitrinib (GA mitochondrial matrix inhibitor), in the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model. The TRAMP mice receiving 3-week or 5-week systemic treatment with Gamitrinib were evaluated for localised or metastatic prostate cancer, prostatic intraepithelial neoplasia (PIN) or localised inflammation using magnetic resonance imaging, histology and immunohistochemistry. Treatment safety was assessed histologically in organs collected at the end of treatment. The effect of Gamitrinib on mitochondrial dysfunction was studied in RM1 cells isolated from TRAMP tumours. RESULTS: Systemic administration of Gamitrinib to TRAMP mice inhibited the formation of localised prostate tumours of neuroendocrine or adenocarcinoma origin, as well as metastatic prostate cancer to abdominal lymph nodes and liver. The Gamitrinib treatment had no effect on PIN or prostatic inflammation, and caused no significant animal weight loss or organ toxicity. Mechanistically, Gamitrinib triggered acute mitochondrial dysfunction in RM1 cells, with loss of organelle inner membrane potential and release of cytochrome-c in the cytosol. CONCLUSIONS: The Gamitrinib has pre-clinical activity and favourable tolerability in a genetic model of localised and metastatic prostate cancer in immunocompetent mice. Selective targeting of mitochondrial Hsp90 could provide novel molecular therapy for patients with advanced prostate cancer.

Regulation of histone gene expression.

Histone genes are expressed during the S phase of the cell cycle. Control is at multiple levels and is mediated by the integration of regulatory signals in response to cell-cycle progression and the onset of differentiation. Much work has been carried out on the H4 gene promoter, which appears to be organized into a series of distinct regulatory elements. The three-dimensional organization of the promoter and, in particular, its spatial relationship with the nuclear matrix scaffold, may be important factors of transcription regulation.

Cells capable of bone production engraft from whole bone marrow transplants in nonablated mice.

Allogeneic and autologous marrow transplants are routinely used to correct a wide variety of diseases. In addition, autologous marrow transplants potentially provide opportune means of delivering genes in transfected, engrafting stem cells. However, relatively little is known about the mechanisms of engraftment in transplant recipients, especially in the nonablated setting and with regard to cells not of hemopoietic origin. In particular, this includes stromal cells and progenitors of the osteoblastic lineage. We have demonstrated for the first time that a whole bone marrow transplant contains cells that engraft and become competent osteoblasts capable of producing bone matrix. This was done at the individual cell level in situ, with significant numbers of donor cells being detected by fluorescence in situ hybridization in whole femoral sections. Engrafted cells were functionally active as osteoblasts producing bone before being encapsulated within the bone lacunae and terminally differentiating into osteocytes. Transplanted cells were also detected as flattened bone lining cells on the periosteal bone surface.

Tumor cells exhibit deregulation of the cell cycle histone gene promoter factor HiNF-D.

Cell cycle-regulated gene expression is essential for normal cell growth and development and loss of stringent growth control is associated with the acquisition of the transformed phenotype. The selective synthesis of histone proteins during the S phase of the cell cycle is required to render cells competent for the ordered packaging of replicating DNA into chromatin. Regulation of H4 histone gene transcription requires the proliferation-specific promoter binding factor HiNF-D. In normal diploid cells, HiNF-D binding activity is regulated during the cell cycle; nuclear protein extracts prepared from normal cells in S phase contain distinct and measurable HiNF-D binding activity, while this activity is barely detectable in G1 phase cells. In contrast, in tumor-derived or transformed cell lines, HiNF-D binding activity is constitutively elevated throughout the cell cycle and declines only with the onset of differentiation. The change from cell cycle-mediated to constitutive interaction of HiNF-D with the promoter of a cell growth-controlled gene is consistent with, and may be functionally related to, the loss of stringent cell growth regulation associated with neoplastic transformation.

Resorption of implanted bone prepared from normal and warfarin-treated rats.

Bone that was virtually depleted of the vitamin K-dependent protein, osteocalcin, and 93% reduced in the concentration of its characteristic amino acid, gamma-carboxyglutamic acid, was obtained from rats treated with warfarin for 6 wk. Osteocalcin-deficient bone particles were resistant to resorption when implanted subcutaneously in normal rats. The relative resorption was 60% of control bone, as measured by histomorphometry as percent of bone particles in the field. Additionally, the number of multinucleated cells around the bone particles was reduced by 54%. These data suggest that osteocalcin is an essential component for bone matrix to elicit progenitor-cell recruitment and differentiation necessary for bone resorption.

Vitamin K-dependent calcium binding proteins in aortic valve calcification.

The pathogenesis of valvar calcification, which complicates the course of cardiac valve disease and also affects tissue valve prostheses, is incompletely understood. The present work explores the possible role of the vitamin K-dependent, calcium-binding amino acid, gamma-carboxyglutamic acid (Gla) in valve mineralization. Gla is normally found in the vitamin K-dependent clotting factor proteins, and is also present in unique calcium binding proteins in bone, kidney, and lung. Unique Gla-containing proteins have also been isolated from pathologic calcifications including calcium containing renal stones and calcified atherosclerotic plaque. Calcified valves including specimens with calcific aortic stenosis, calcified porcine xenograft valves, and a calcified aortic homograft valve were analyzed for Gla content, complete amino acid analysis, and tissue calcium and phosphorus levels. Normal porcine valves contained protein-bound Gla (2.0-10.6 Gla/10(4) amino acids): no Gla was present in normal valve leaflets. Furthermore, Gla levels paralleled tissue calcium content in the calcified valves. In addition, complete amino acid analysis indicated a decline in valvar collagen content plus increased acidic proteins in conjunction with valvar calcification and the presence of Gla-containing proteins. These results suggest that calcific valvar disease may result in part from vitamin K-dependent processes.

The presence of protein-bound gamma-carboxyglutamic acid in calcium-containing renal calculi.

The amino acid gamma-carboxyglutamic acid (Gla) is found in four blood-clotting proteins, in a bone protein, in kidney protein, and in the protein present in various ectopic calcifications. This paper reports the presence of Gla in the EDTA-soluble, nondialyzable proteins of calcium-containing renal calculi including calcium oxalate, hydroxyapatite, and mixed stores of apatite and struvite (MgNH4PO4). Calculi composed of pure struvite and those composed of only uric acid or cystine do not contain Gla. From calcium oxalate and hydroxyapatite stontes, a protein of about 17,000 daltons was obtained which contained about 40 residues of Gla per 1,000 amino acids. The amino acid composition of this protein had no apparent relationship to the Gla-containing bone protein or to the similarly-sized F1 fragment of prothrombin which contains about 64 residues of Gla per 1,000 amino acid residues. The Gla-rich protein in calcium-containing renal stones thus may be a different Gla-containing protein. These data as well as other studies demonstrating the presence of Gla in pathologically calcified tissues not normally containing Gla suggest that the Gla-containing proteins may be of considerable pathophysiological significance.

Overlapping and CpG methylation-sensitive protein-DNA interactions at the histone H4 transcriptional cell cycle domain: distinctions between two human H4 gene promoters.

Transcriptional regulation of vertebrate histone genes during the cell cycle is mediated by several factors interacting with a series of cis-acting elements located in the 5' regions of these genes. The arrangement of these promoter elements is different for each gene. However, most histone H4 gene promoters contain a highly conserved sequence immediately upstream of the TATA box (H4 subtype consensus sequence), and this region in the human H4 gene FO108 is involved in cell cycle control. The sequence-specific interaction of nuclear factor HiNF-D with this key proximal promoter element of the H4-FO108 gene is cell cycle regulated in normal diploid cells (J. Holthuis, T.A. Owen, A.J. van Wijnen, K.L. Wright, A. Ramsey-Ewing, M.B. Kennedy, R. Carter, S.C. Cosenza, K.J. Soprano, J.B. Lian, J.L. Stein, and G.S. Stein, Science, 247:1454-1457, 1990). Here, we show that this region of the H4-FO108 gene represents a composite protein-DNA interaction domain for several distinct sequence-specific DNA-binding activities, including HiNF-D, HiNF-M, and HiNF-P. Factor HiNF-P is similar to H4TF-2, a DNA-binding activity that is not cell cycle regulated and that interacts with the analogous region of the H4 gene H4.A (F. LaBella and N. Heintz, Mol. Cell. Biol. 11:5825-5831, 1991). The H4.A gene fails to interact with factors HiNF-M and HiNF-D owing to two independent sets of specific nucleotide variants, indicating differences in protein-DNA interactions between these H4 genes. Cytosine methylation of a highly conserved CpG dinucleotide interferes with binding of HiNF-P/H4TF-2 to both the H4-FO108 and H4.A promoters, but no effect is observed for either HiNF-M or HiNF-D binding to the H4-FO108 gene. Thus, strong evolutionary conservation of the H4 consensus sequence may be related to combinatorial interactions involving overlapping and interdigitated recognition nucleotides for several proteins, whose activities are regulated independently. Our results also suggest molecular complexity in the transcriptional regulation of distinct human H4 genes.

runt homology domain transcription factors (Runx, Cbfa, and AML) mediate repression of the bone sialoprotein promoter: evidence for promoter context-dependent activity of Cbfa proteins.

Expression of the bone sialoprotein (BSP) gene, a marker of bone formation, is largely restricted to cells in mineralized tissues. Recent studies have shown that the Cbfa1 (also known as Runx2, AML-3, and PEBP2alphaA) transcription factor supports commitment and differentiation of progenitor cells to hypertrophic chondrocytes and osteoblasts. This study addresses the functional involvement of Cbfa sites in expression of the Gallus BSP gene. Gel mobility shift analyses with nuclear extracts from ROS 17/2.8 osteoblastic cells revealed that multiple Cbfa consensus sequences are functional Cbfa DNA binding sites. Responsiveness of the 1.2-kb Gallus BSP promoter to Cbfa factors Cbfa1, Cbfa2, and Cbfa3 was assayed in osseous and nonosseous cells. Each of the Cbfa factors mediated repression of the wild-type BSP promoter, in contrast to their well known activation of various hematopoietic and skeletal phenotypic genes. Suppression of BSP by Cbfa factors was not observed in BSP promoters in which Cbfa sites were deleted or mutated. Expression of the endogenous BSP gene in Gallus osteoblasts was similarly downregulated by forced expression of Cbfa factors. Our data indicate that Cbfa repression of the BSP promoter does not involve the transducin-like enhancer (TLE) proteins. Neither coexpression of TLE1 or TLE2 nor the absence of the TLE interaction motif of Cbfa1 (amino acids 501 to 513) influenced repressor activity. However, removal of the C terminus of Cbfa1 (amino acids 362 to 513) relieved suppression of the BSP promoter. Our results, together with the evolutionary conservation of the seven Cbfa sites in the Gallus and human BSP promoters, suggest that suppressor activity by Cbfa is of significant physiologic consequence and may contribute to spatiotemporal expression of BSP during bone development.

Multiple Cbfa/AML sites in the rat osteocalcin promoter are required for basal and vitamin D-responsive transcription and contribute to chromatin organization.

Three Cbfa motifs are strategically positioned in the bone-specific rat osteocalcin (rOC) promoter. Sites A and B flank the vitamin D response element in the distal promoter and sites B and C flank a positioned nucleosome in the proximal promoter. The functional significance of each Cbfa element was addressed by mutating individual or multiple Cbfa sites within the context of the -1.1-kb rOC promoter fused to a chloramphenicol acetyltransferase reporter gene. Promoter activity was assayed following transient transfection and after stable genomic integration in ROS 17/2.8 osteoblastic cell lines. We show that all three Cbfa sites are required for maximal basal expression of the rOC promoter. However, the distal sites A and B each contribute significantly more (P < 0.001) to promoter activity than site C. In a genomic context, sites A and B can largely compensate for a mutation at the proximal site C, and paired mutations involving site A (mAB or mAC) result in a far greater loss of activity than the mBC mutation. Strikingly, mutation of the three Cbfa sites leads to abrogation of responsiveness to vitamin D. Vitamin D-enhanced activity is also not observed when sites A and B are mutated. Significantly, related to these losses in transcriptional activity, mutation of the three Cbfa sites results in altered chromatin structure as reflected by loss of DNase I-hypersensitive sites at the vitamin D response element and over the proximal tissue-specific basal promoter. These findings strongly support a multifunctional role for Cbfa factors in regulating gene expression, not only as simple transcriptional transactivators but also by facilitating modifications in promoter architecture and chromatin organization.

Replication-dependent histone gene expression is related to Cajal body (CB) association but does not require sustained CB contact.

Interactions between Cajal bodies (CBs) and replication-dependent histone loci occur more frequently than for other mRNA-encoding genes, but such interactions are not seen with all alleles at a given time. Because CBs contain factors required for transcriptional regulation and 3' end processing of nonpolyadenylated replication-dependent histone transcripts, we investigated whether interaction with CBs is related to metabolism of these transcripts, known to vary during the cell cycle. Our experiments revealed that a locus containing a cell cycle-independent, replacement histone gene that produces polyadenylated transcripts does not preferentially associate with CBs. Furthermore, modest but significant changes in association levels of CBs with replication-dependent histone loci mimic their cell cycle modulations in transcription and 3' end processing rates. By simultaneously visualizing replication-dependent histone genes and their nuclear transcripts for the first time, we surprisingly find that the vast majority of loci producing detectable RNA foci do not contact CBs. These studies suggest some link between CB association and unusual features of replication-dependent histone gene expression. However, sustained CB contact is not a requirement for their expression, consistent with our observations of U7 snRNP distributions. The modest correlation to gene expression instead may reflect transient gene signaling or the nucleation of small CBs at gene loci.

Nuclear structure-gene expression interrelationships: implications for aberrant gene expression in cancer.

There is long-standing recognition that transformed and tumor cells exhibit striking alterations in nuclear morphology as well as in the representation and intranuclear distribution of nucleic acids and regulatory factors. Parameters of nuclear structure support cell growth and phenotypic properties of cells by facilitating the organization of genes, replication and transcription sites, chromatin remodeling complexes, transcripts, and regulatory factors in structurally and functionally definable subnuclear domains within the three-dimensional context of nuclear architecture. The emerging evidence for functional interrelationships of nuclear structure and gene expression is consistent with linkage of tumor-related modifications in nuclear organization to compromised gene regulation during the onset and progression of cancer.

Expression of cell cycle regulatory factors in differentiating osteoblasts: postproliferative up-regulation of cyclins B and E.

The representation of cyclins and cyclin-dependent kinases (cdks) was analyzed during progressive development of the bone cell phenotype in cultures of normal diploid rat calvarial osteoblasts. Three developmental stages were examined: (a) proliferation; (b) monolayer confluency; and (c) mineralization of the bone extracellular matrix. We demonstrate that the presence of cyclins and cdks is not restricted to the proliferation period. Consistent with their role in cell cycle progression, cdc2 and cdk2 decrease postproliferatively. However, cdk4 and cyclins A, B, and D1 persist in confluent cells. Cyclin E is significantly up-regulated during the extracellular matrix mineralization developmental period. Examination of the cytoplasmic levels of these cell cycle regulatory proteins indicates a marked increase in cyclin B in the late differentiation stage. The elevation of nuclear cyclin E and cytoplasmic cyclin B is not observed in osteoblasts maintained under culture conditions that do not support differentiation. Furthermore, treatment with transforming growth factor beta for 48 h during the proliferation period renders the cells incompetent for differentiation and abrogates the postproliferative up-regulation of cyclins B and E. Density-induced growth inhibition of ROS 17/2.8 osteosarcoma cells is not accompanied by up-regulation of nuclear cyclin E and cytoplasmic cyclin B when compared to the proliferation period. This observation is consistent with abrogation of both growth control and differentiation regulatory mechanisms in tumor cells. These results suggest that cell cycle regulatory proteins function not only during proliferation but may also play a role in normal diploid osteoblast differentiation.

Nuclear matrix proteins distinguish normal diploid osteoblasts from osteosarcoma cells.

Interrelationships between nuclear architecture and gene expression were examined by comparing the representation of nuclear matrix proteins in ROS 17/2.8 rat and MG-63 human osteosarcoma cells with those in normal diploid osteoblasts. The tumor-derived cells coexpress genes which are expressed in a sequential and mutually exclusive manner during the progressive stages of osteoblast differentiation. In osteosarcoma cells two-dimensional electrophoretic analysis indicates a composite representation of nuclear matrix proteins characteristic of both the proliferative and postproliferative periods of osteoblast phenotype development. In addition, nuclear matrix proteins unique to the tumor cells and the absence of nuclear matrix proteins found only in normal diploid osteoblasts are observed. Tumor-specific nuclear matrix proteins include those expressed in a proliferation-dependent and independent manner. There is a parallel relationship between nuclear matrix proteins and the expression of cell growth and tissue-specific genes during osteoblast differentiation and in osteosarcoma cells where the developmental sequence of gene expression has been abrogated. Nuclear matrix proteins therefore provide markers reflecting defined periods of bone cell differentiation and phenotypic characteristics of an osteosarcoma.

cis-diamminedichloroplatinum(II) cross-linking of the human myeloid cell nuclear differentiation antigen to DNA in HL-60 cells following 1,25-dihydroxy vitamin D3-induced monocyte differentiation.

Protein-DNA interactions of the human myeloid cell nuclear differentiation antigen (MNDA) were examined in vivo in proliferating HL-60 promyelocytic leukemia cells and following induction of differentiation by 1,25-dehydroxyvitamin D3. Intact cells were treated with the reversible cross-linking agent cis-diamminedichloroplatinum(II) and the MNDA levels in the isolated protein-DNA complexes were determined. Less than 1% of the total intracellular level of MNDA was cross-linked to DNA in the noninduced proliferating HL-60 cells. Once the cells were induced to differentiate into monocytes, the amount of antigen cross-linked to the DNA increased to over 5% of the total intracellular level. The increased efficiency of cross-linking the MNDA to DNA was specific for monocyte-induced HL-60 differentiation, achieved with three inducers, and was not observed in association with granulocyte-induced differentiation. On a molar basis the phorbol ester (12-O-tetradecanoylphorbol-13-acetate) was the most effective inducer of monocyte differentiation, followed by 1,25-dihydroxy-16-ene-23-ynecholecalciferol which was more effective than 1,25-dihydroxycholecalciferol. A cesium chloride gradient analysis of the nucleic acid-protein fraction isolation from cis-diamminedichloroplatinum(II)-treated, monocyte-induced HL-60 cells documented the authenticity of the association between the MNDA and DNA. The results indicate that a significant level of chromatin reorganization may accompany monocyte-induced differentiation that leads to much higher levels of MNDA-DNA cross-linking to DNA. The expression of the MNDA is restricted to human myeloid cells and the present results indicate that a fraction of this low abundance nuclear protein is specifically located near the DNA [within cis-diamminedichloroplatinum(II) cross-linking distance] and that this association may be modulated specifically during monocyte differentiation.

The cell cycle regulator p27kip1 contributes to growth and differentiation of osteoblasts.

The cyclin-dependent kinase (cdk) inhibitors are key regulators of cell cycle progression. p27 and p21 are members of the Cip/Kip family of cdk inhibitors and regulate cell growth by inactivating cell cycle stage-specific CDK-cyclin complexes. Because down-regulation of osteoprogenitor proliferation is a critical step for osteoblast differentiation, we investigated expression of p27 and p21 during development of the osteoblast phenotype in rat calvarial osteoblasts and in proliferating and growth-inhibited osteosarcoma ROS 17/2.8 cells. Expression of these proteins indicates that p21, which predominates in the growth period, is related to proliferation control. p27 levels are maximal postproliferatively, suggesting a role in the transition from cell proliferation to osteoblast differentiation. We directly examined the role of p27 during differentiation of osteoprogenitor cells derived from the bone marrow (BM) of p27-/- mice. BM cells from p27 null mice exhibited increased proliferative activity compared with BM cells from wild-type mice and formed an increased number and larger size of osteoblastic colonies, which further differentiated to the mineralization stage. Although p27-/- adherent marrow cells proliferate faster, they retain competency for differentiation, which may result, in part, from observed higher p21 levels compared with wild type. Histological studies of p27-/- bones also showed an increased cellularity in the marrow cavity compared with the p27+/+. The increased proliferation in bone does not lead to tumorigenesis, in contrast to observed adenomas in the null mice. Taken together, these findings indicate that p27 plays a key role in regulating osteoblast differentiation by controlling proliferation-related events in bone cells.