A potential dichotomous role of ATF3, an adaptive-response gene, in cancer development.

Activating transcription factor 3 (ATF3) is a member of the ATF/cyclic AMP response element-binding family of transcription factors. We present evidence that ATF3 has a dichotomous role in cancer development. By both gain- and loss-of-function approaches, we found that ATF3 enhances apoptosis in the untransformed MCF10A mammary epithelial cells, but protects the aggressive MCF10CA1a cells and enhances its cell motility. Array analyses indicated that ATF3 upregulates the expression of several genes in the tumor necrosis factor pathway in the MCF10A cells but upregulates the expression of several genes implicated in tumor metastasis, including TWIST1, fibronectin (FN)-1, plasminogen activator inhibitor-1, urokinase-type plasminogen activator, caveolin-1 and Slug, in the MCF10CA1a cells. We present evidence that ATF3 binds to the endogenous promoters and regulates the transcription of the TWIST1, FN-1, Snail and Slug genes. Furthermore, conditioned medium experiments indicated that ATF3 has a paracrine/autocrine effect, consistent with its upregulation of genes encoding secreted factors. Finally, ATF3 gene copy number is >2 in approximately 80% of the breast tumors examined (N=48) and its protein level is elevated in approximately 50% of the tumors. These results provided a correlative argument that it is advantageous for the malignant cancer cells to express ATF3, consistent with its oncogenic roles suggested by the MCF10CA1a cell data.

Promoter methylation reduces C/EBPdelta (CEBPD) gene expression in the SUM-52PE human breast cancer cell line and in primary breast tumors.

CCAAT/Enhancer Binding Proteins (C/EBPs) are a highly conserved family of leucine zipper proteins that regulate cell growth and differentiation. C/EBPdelta functions in the initiation and maintenance of mammary epithelial cell G(0) growth arrest and 'loss of function' alterations in C/EBPdelta gene expression have been reported in human breast cancer and in rodent carcinogen-induced mammary tumors. The molecular mechanism underlying reduced C/EBPdelta gene expression in mammary tumorigenesis, however, is unknown. In this report we demonstrate that C/EBPdelta gene expression is undetectable in the SUM-52PE human breast cancer cell line and that silencing of SUM-52PE C/EBPdelta gene expression is due to epigenetic promoter hypermethylation (26/27 CpGs methylated). The hypermethylated SUM-52PE C/EBPdelta gene promoter is associated with reduced levels of acetylated Histone H4, consistent with a closed, transcriptionally inactive chromatin conformation. Treatment with 5'-aza-cytidine and trichostatin A (TSA) re-activates cytokine-induced SUM-52PE C/EBPdelta gene expression. C/EBPdelta gene expression is reduced to virtually undetectable levels in 32% (18/57) of primary human breast tumors. Site-specific CpG methylation was observed in 33% (6/18) of the low C/EBPdelta expressing primary breast tumors. CpG methylation adjacent to the C/EBPdelta proximal promoter Sp1 site was associated with reduced C/EBPdelta expression in a primary breast cancer sample. Electromobility shift assays (EMSA) demonstrated a significant reduction in binding to oligos containing the CpG methylation 5' to the Sp1 binding site. These results demonstrate a direct link between C/EBPdelta gene promoter hyper- and site specific-methylation and reduced C/EBPdelta gene expression in breast cancer cell lines and primary breast tumors.

CCAAT/Enhancer binding protein delta (c/EBPdelta) regulation and expression in human mammary epithelial cells: II. Analysis of activating signal transduction pathways, transcriptional, post-transcriptional, and post-translational control.

CCAAT/enhancer binding protein delta (C/EBPdelta) plays a key role in mammary epithelial cell G0 growth arrest. C/EBPdelta gene expression is down-regulated in rodent mammary tumorigenesis and in human breast cancer, suggesting that "loss of function" alterations in C/EBPdelta gene expression are common in mammary gland malignancies. The goal of this study was to systematically investigate the mechanisms controlling C/EBPdelta gene expression in MCF-10A and MCF-12A human mammary epithelial cell lines. The results demonstrate that G0 growth arrest conditions (i.e., serum and growth factor withdrawal or Oncostatin M (OSM) treatment) result in the activation of JAK1, JAK2, and Tyk 2, members of the Janus kinase family of non-receptor tyrosine kinases, in MCF-10A and MCF-12A cells. Growth arrest or OSM treatment also specifically increases activated (phosphorylated) signal transduction and activators of transcription 3 (STAT3) levels, demonstrating that STAT3, not STAT1 or STAT5, is the downstream target of the activated Janus kinases in MCF-10A and MCF-12A cells. Whole cell lysates from G0 growth arrested (GA) and OSM-treated MCF-12A cells exhibit increased acute phase response element (APRE) binding compared to lysates from growing (GR) MCF-12A cells. Transient transfection using C/EBPdelta promoter-luciferase constructs demonstrated that the APRE (STAT3) consensus binding site is essential for growth arrest or OSM induction of the C/EBPdelta promoter. Mutation of the C/EBPdelta promoter STAT3 site or expression of a dominant negative STAT3 construct (STAT3delta) reduces C/EBPdelta promoter activity in response to growth arrest conditions. The human C/EBPdelta promoter also contains an Sp1 site at -61 bp (relative to the transcriptional start site) which is required for basal transcriptional activation. Mutation or deletion of the Sp1 site decreases promoter activity in response to growth arrest conditions. Treatment with the transcriptional inhibitor actinomycin D demonstrated that the C/EBPdelta mRNA exhibits a relatively short half-life (approximately 40 min). Similarly, treatment with the translational inhibitor anisomysin demonstrated that the C/EBPdelta protein half-life was also relatively short (approximately 160 min). These results indicate that the human C/EBPdelta gene is controlled at multiple levels, consistent with a role for C/EBPdelta in cell cycle control and/or cell fate determination.

CCAAT/Enhancer binding protein delta (c/EBPdelta) regulation and expression in human mammary epithelial cells: I. "Loss of function" alterations in the c/EBPdelta growth inhibitory pathway in breast cancer cell lines.

"Loss of function" alterations in growth inhibitory signal transduction pathways are common in cancer cells. In this study, we show that growth arrest (GA) treatments--serum and growth factor withdrawal and growth inhibitory IL-6 family cytokines (Interleukin-6 and Oncostatin M (OSM))--increase STAT3 phosphorylation (pSTAT3), increase CCAAT enhancer binding protein delta (C/EBPdelta) gene expression and induce GA of primary, finite-lifespan human mammary epithelial cells (HMECs), and immortalized breast cell lines (MCF-10A and MCF-12A). In contrast, serum and growth factor withdrawal from human breast cancer cell lines (MCF-7, SK-BR-3, T-47D, and MDA-MB-231) for up to 48 h induced a relatively modest increase in pSTAT3 levels and C/EBPdelta gene expression and resulted in varying levels of GA. In most breast cancer cell lines, IL-6 family cytokine treatment increased pSTAT3 levels and C/EBPdelta gene expression, however, growth inhibition was cell line dependent. In addition to "loss of function" alterations in growth inhibitory pathways, breast cancer cell lines also exhibit "gain of function" alterations in growth signaling pathways. The Akt growth/ survival pathway is constitutively activated in T-47D and MCF-7 breast cancer cells. The Akt inhibitor LY 294,002 significantly enhanced T-47D growth inhibition by serum and growth factor withdrawal or IL-6 family cytokine treatment. Finally, we show that activation of the pSTAT3/C/EBPdelta growth control pathway is independent of estrogen receptor status. These results demonstrate that "loss of function" alterations in the pSTAT3/C/EBPdelta growth inhibitory signal transduction pathway are relatively common in human breast cancer cell lines. Defective activation of the pSTAT3/ C/EBPdelta growth inhibitory signal transduction pathway, in conjunction with constitutive activation of the Akt growth stimulatory pathway, may play a synergistic role in the etiology or progression of breast cancer.

Local signals induce CCAAT/enhancer binding protein-delta (C/EBP-delta) and C/EBP-beta mRNA expression in the involuting mouse mammary gland.

The repeated lactation cycles in the mammary gland offer a unique environment for the study of cell growth, differentiation, and death. The CCAAT/enhancer binding proteins (C/EBPs) are a family of transcription factors important in growth control and differentiation in many tissues. Our laboratory and others have shown that C/EBP-delta and C/EBP-beta mRNA expression is closely associated with normal mouse mammary gland involution. To examine the relative influence of local versus systemic factors in C/EBP expression and tissue remodeling, a gland sealing mouse model was used. Mice with unilateral sealing continue to lactate and nurse pups via nonsealed glands, while sealed glands initiate involution. The expression of C/EBP-alpha, beta and delta mRNA was investigated in sealed and nonsealed nursing glands. In situ apoptosis was documented and glandular morphology was also examined. C/EBP-delta mRNA levels are low in nonsealed glands, but are rapidly and transiently induced in sealed glands by 24 h. C/EBP-beta mRNA expression is also relatively low in nonsealed glands, but is induced in sealed glands within 72 h. Expression of the apoptosis-associated mRNAs encoding bax and TRPM-2 is also induced in sealed glands by 24-48 h. Apoptosis and a moderate degree of tissue remodeling occur within the sealed glands in spite of systemic hormone levels capable of sustaining lactation. These data demonstrate that local factors are sufficient to induce C/EBP-beta and C/EBP-delta in the mouse mammary gland. In addition, mammary epithelial apoptosis and glandular remodeling occur in sealed glands, confirming a critical role for local factors in mammary involution.

Lactation status influences expression of CCAAT/enhancer binding protein isoform mRNA in the mouse mammary gland.

The CCAAT/enhancer binding proteins (C/EBPs) are a highly conserved family of DNA binding proteins implicated in the transcriptional control of genes involved in cell growth and differentiation in a variety of tissues. The expression of C/EBP-alpha, beta, and delta mRNA in the normal mouse mammary gland was investigated during pregnancy, lactation, and involution via Northern blotting and in situ hybridization. Mammary gland C/EBP-alpha mRNA is detectable at low levels during pregnancy and postlactational involution. C/EBP-beta mRNA levels are elevated during pregnancy, decline slightly in midlactation, and are induced within 48 hours of the onset of involution. C/EBP-delta mRNA content is low throughout pregnancy and lactation, but increases dramatically (>100-fold) within 12 hours after the onset of postlactational involution. In situ hybridization demonstrates that mammary epithelial cells are responsible for the expression of C/EBP-delta mRNA during involution. In contrast to mammary gland, C/EBP-alpha is the predominate isoform expressed in liver with relatively low expression of C/EBP-beta and C/EBP-delta mRNA. Liver C/EBP isoform mRNA levels are unaffected by lactation status. These results demonstrate the tissue-specific regulation of C/EBPs. The pronounced sequential induction of C/EBP-delta and C/EBP-beta during postlactational involution is consistent with a role for C/EBPs in the regulation of mammary epithelial cell apoptosis.

Sequences of the cDNAs encoding canine parathyroid hormone-related protein and parathyroid hormone.

The cDNA clones encoding canine parathyroid-hormone-related protein (cPTHrP) and parathyroid hormone (cPTH) have been isolated and sequenced. The predicted amino-acid sequences of the mature canine homologs have a high degree of homology to human PTHrP (hPTHrP) and PTH (hPTH), especially in the biologically active regions. The cPTHrP cDNA is unique, since it has homology to exon 1A of hPTHrP which suggests that dogs utilize a promoter similar to P1 of hPTHrP which has not been demonstrated in other species.

Effects of transforming growth factor-beta on parathyroid hormone-related protein production and ribonucleic acid expression by a squamous carcinoma cell line in vitro.

The effects of human recombinant transforming growth factor (TGF)-beta 1 were determined on PTH-related protein (PTHrP) production and messenger RNA (mRNA) expression by a canine squamous carcinoma cell line (SCC 2/88) in vitro. TGF-beta increased PTHrP production in a dose- and time-dependent manner (P < 0.05) as measured by RIA, and the effects of TGF-beta treatment persisted up to 72 h after removal. TGF-beta increased PTHrP production by SCC 2/88 cells until cellular confluence, at which time there was no longer a significant increase compared to control. Actinomycin D inhibited the TGF-beta-mediated increase in PTHrP production, suggesting that TGF-beta acted in part by increasing gene transcription. SCC 2/88 cells also produced active TGF-beta as measured by a [3H]thymidine incorporation assay in mink lung epithelial cells. Exposure of SCC 2/88 cells to a neutralizing anti-TGF-beta monoclonal antibody decreased (up to 50%, P < 0.01) basal PTHrP production. TGF-beta increased PTHrP mRNA expression in a dose- and time-dependent manner as evaluated by northern blot analysis. Postconfluent SCC 2/88 cells expressed little mRNA for PTHrP, and there was only a minimal increase in PTHrP mRNA expression in postconfluent TGF-beta-treated cells. These results indicate that TGF-beta increased PTHrP production and mRNA expression in malignant keratinocytes and suggest that TGF-beta may be an important factor in the pathogenesis of humoral hypercalcemia of malignancy.

Structural analysis of the ras transgene in MMTV/v-Ha-ras transgenic mice.

Transgenic animals are becoming increasingly important in laboratory animal research. In cancer research, the role of specific genes in tumorigenesis can be directly tested in live animals by using transgenic animal technology. Since DNA-damaging carcinogens are not required to initiate tumors in transgenic animals, these models are particularly useful in the analysis of genetic alterations associated with tumorigenesis. Southern blot analysis was used to assess the copy number, structure, and methylation status of the ras trans-gene in MMTV/v-Ha-ras transgenic mice. The results indicate that MMTV/v-Ha-ras transgenic mice carry about 20 copies of the ras transgene. The integrated ras transgene is maintained without major rearrangements in normal tissues and in mammary tumors. The ras transgene is methylated in liver, hypomethylated at a single site in normal mammary tissue, and hypomethylated at two sites in mammary tumors. Transgenic animals provide a new model to assess genetic alteration in tumorigenesis.

Linoleic acid controls neonatal tissue-specific stearoyl-CoA desaturase mRNA levels.

The mouse genome contains two stearoyl-CoA desaturase (SCD) structural genes (SCD1 and SCD2) that are expressed in a tissue-specific manner. Brain SCD2 mRNA levels are about 2-fold higher in pups nursed by mothers fed a control diet (5% corn oil (CO), (essential fatty acid (EFA) adequate)), compared with brain SCD2 mRNA levels in pups nursed by mothers fed an EFA-deficient (EFAD) diet (5% coconut oil (COCO)). In contrast to brain, control pup hepatic SCD1 mRNA levels are reduced to < 1.0% of the EFAD pup hepatic SCD1 mRNA levels. EFA status does not alter SCD1 or SCD2 transcription initiation sites. CCAAT/enhancer-binding proteins (C/EBP) have been implicated in the transcriptional control of key genes in energy metabolism. Both the SCD1 and SCD2 gene promoters contain C/EBP transcription factor consensus-binding sites. Neonatal mouse liver expresses C/EBP-alpha, C/EBP-beta and C/EBP-delta mRNAs. In contrast neonatal mouse brain expresses high levels of C/EBP-delta, but little C/EBP-alpha or C/EBP-beta mRNA. EFA intake has no effect on tissue-specific C/EBP isoform mRNA levels suggesting that C/EBP isoform function is controlled at the translational or post-translational level.

Dietary fat promotes mammary tumorigenesis in MMTV/v-Ha-ras transgenic mice.

The purpose of this study was to investigate the influence of dietary fat on mammary tumorigenesis in MMTV/v-Ha-ras transgenic mice. Female MMTV/v-Ha-ras transgenics were fed diets providing 0, 5 or 25% of calories from corn oil (CO). The mammary tumor incidence was 7% (0% CO), 36% (5% CO) and 52% (25% CO). Ras mRNA levels were increased in mammary tumors in the 25% CO group. The ras transgene was hypomethylated in mammary tumors, but not in liver or nontransformed mammary tissue. Mammary tumors expressed apolipoprotein E mRNA. Alterations in gene structure and expression in transgenic mice may suggest mechanisms by which dietary fat promotes mammary tumors.

Quaking phenotype influences brain lipid-related mRNA levels.

Although lipids compose almost 80% of myelin, the influence of quaking on mRNAs encoding lipid biosynthetic enzymes and transport proteins has not been previously reported. Understanding the influence of quaking on myelin-specific and lipid-related mRNAs will be useful in determining the mechanism of the quaking defect. Stearoyl CoA desaturase (SCD) catalyzes a key step in the biosynthesis of oleic acid (C18:1, n-9), a major fatty acid in myelin. SCD, LDL receptor (LDLR) and apolipoprotein E (Apo E) mRNA levels are all reduced in neonatal quaking brains. In contrast to brain, quaking hepatic LDLR and Apo E mRNA levels are normal. These results indicate that lipid-related mRNAs are reduced in neonatal quaking brain, but the quaking liver is unaffected. The quaking defect influences gene expression in multiple cell types of glial lineage in the developing CNS.

Postnatal dietary fat influences mRNAS involved in myelination.

The synthesis and composition of myelin in the developing mouse central nervous system can be influenced by diet. Postnatal maternal fat intake altered nursing pup brain and liver fatty acid composition. Peak (day 21) proteolipid protein (PLP) and myelin basic protein (MBP) mRNA levels were reduced when pups were nursed by mothers fed a fat-free or 5% coconut oil diet. This effect was reversed by feeding a corn oil based diet. Oleic acid accounts for about 30% of myelin fatty acids. mRNA levels of stearoyl CoA desaturase (SCD), the rate-limiting step in oleic acid synthesis, increase in neonatal mouse brain. Postnatal maternal fat-free feeding reduced day 21 pup brain SCD and LDL receptor, but not apolipoprotein (Apo E) E mRNA levels. In contrast to brain, nursing pup hepatic SCD mRNA levels were induced, LDL receptor mRNA levels were unaffected and Apo E mRNA levels were reduced by postnatal maternal fat-free feeding. Myelin-specific mRNA levels are developmentally regulated and influenced by dietary fat. Neonatal brain SCD and LDL receptor mRNA levels are also altered by neonatal fat intake. The neonatal response to dietary fat is tissue-specific at the mRNA level.

Analysis of the multiple 5' and 3' termini of poly(A)+ and poly(A)-deficient thymidylate synthase mRNA in growth-stimulated mouse fibroblasts.

Thymidylate synthase (TS) mRNA content increases about 20-fold when growth-stimulated mouse cells progress from the G0/G1 phase into the S phase of the cell cycle. Previous studies, using a cell line in which the TS gene is amplified (LU3-7), indicated that transcriptional initiation as well as polyadenylation of the mRNA occur at several locations in unsynchronized cells. In the present study, we have used S1 nuclease protection assays to analyze the possible significance of the multiple transcriptional initiation and polyadenylation sites. We found that the same pattern of 5' and 3' termini were detected with RNA isolated from the overproducing cells as with RNA isolated from the parental mouse 3T6 cell line, demonstrating that the heterogeneous termini are not a consequence of gene amplification. There was no change in the pattern of 5' or 3' termini with either cell line during the progression from G1 phase through S phase in serum-stimulated cells. Therefore, the increase in TS mRNA content is not the result of differential utilization of the various transcriptional initiation or polyadenylation sites. Analyses of poly(A)- deficient cytoplasmic TS RNA showed that the 5' termini were the same as those found in poly(A) + mRNA. However, the 3' termini were extremely heterogeneous in length. Although some of the poly(A)- deficient RNA extended beyond the normal site of polyadenylation, most of it was shorter than full-length TS mRNA.

Construction and expression of mouse thymidylate synthase minigenes.

Mouse thymidylate synthase minigenes that lack introns were constructed by ligating restriction fragments containing 4.5, 1.0, or 0.25 kilobase pairs (kb) of 5'-flanking DNA of the normal thymidylate synthase gene and as little as 0.25 kb of 3'-flanking DNA to full-length thymidylate synthase cDNA. All three minigenes were expressed at approximately the same levels following transfection into hamster V79 cells that were deficient in thymidylate synthase. S1 nuclease protection assays revealed that the multiple 5' and 3' termini of thymidylate synthase mRNA in cells transfected with these minigenes were at the same positions as those of the normal mRNA in mouse cells. Deletion analysis of the promoter region revealed that minigenes extending to position -150 nucleotides (relative to the AUG codon) were expressed at approximately the same level as those extending to -1 kb. However, minigenes extending to -53 nucleotides were inactive. To determine if the minigenes were capable of being regulated in a cell cycle-dependent manner, thymidylate synthase gene expression was measured in hamster cells that were stably transfected with the largest minigene and synchronized by serum-stimulation. Thymidylate synthase enzyme level and mRNA content increased 3-5-fold as cells progressed from G1 through S phase.

Effects of dietary fatty acids on delayed-type hypersensitivity in mice.

Effects of an essential fatty acid deficient (EFAD) [0% corn oil (CO)] diet and a diet high in polyunsaturated fatty acids [PUFA (50% CO)] on one aspect of in vivo T cell function [delayed-type hypersensitivity (DTH)] were assessed. After a 70-day feeding trial, DTH was reduced by 30% in mice fed the EFAD diet, but the response of mice fed the high PUFA diet equaled that of control mice fed a diet containing 13% CO. The time required for the EFAD diet to reduce DTH was 42 days. Although consumption of the EFAD diet reduced DTH, this reduction was rapidly reversed, within 7 days, by switching the EFAD mice to the control diet. These results indicate that :1) consumption of the EFAD diet reduces one aspect of in vivo T cell function (DTH), but the effect can be reversed by refeeding the control diet; and 2) a high PUFA diet does not adversely affect DTH.

Effects of essential fatty acid deficiency, and various levels of dietary polyunsaturated fatty acids, on humoral immunity in mice.

Six experiments were conducted to determine the influence of an essential fatty acid deficient diet (EFAD), and various levels of dietary polyunsaturated fatty acids, on humoral immunity in mice. The results indicated that: 1) Consumption of diets deficient in essential fatty acids (0% corn oil) significantly reduced the humoral response. This reduction was demonstrated after feeding the essential fatty acid deficient diet for only 28 days; and preceded the effects of essential fatty acid deficiency on growth or appearance. 2) Reduced antibody response was demonstrated against T-cell dependent and T-cell independent antigens, and in both primary and secondary responses of mice fed the essential fatty acid deficient diet. 3) After 56 days of feeding the EFAD diet (0% corn oil), mice switched to the control diet (13% corn oil) for 7 days demonstrated full recovery of the humoral response. 4) Diets containing various levels of polyunsaturated fatty acids (from 2 to 70% of energy from corn oil) did not adversely affect the humoral response. The results support the hypothesis that essential fatty acids play a crucial role in maintaining the functional integrity of humoral immunity.