Our previous experimental results showed that Z stimulates MCF-7 cell and MCF-10A cell growth [10]. The serum harvested from the heifer one month post Z implantation is more potent in stimulating human breast cancer cell lines as well as PCHBCEC growth than that from control heifers. In the current study, we evaluated the effects of Z on PCHNBEC’s and PCHBCEC’s growth. The results show Z increased the proliferation of both PCHNBECs and PCHBCECs in a dose-dependent manner (Figure 1). PCHBCECs are more sensitive in response to Z treatment than PCHNBECs (Figure 1).

In order to explore the possible mechanisms involved in Z stimulation of PCHNBECs and PCHBCECs proliferation, we examined the expression of the p53 gene at the mRNA and proteins level. p53 is involved in many important physiological processes such as cell cycle arrest, gene transcription, DNA repair, and apoptosis. Our result illustrated that Z treatment significantly suppressed the level of p53 mRNA and protein expression in PCHBCECs in comparison to the levels in the control group (Figures 2 and 4). However, Z did not show significant reduction of p53 mRNA and protein expression in PCHNBECs when compared to the control (Figure 2).

DNMT1 plays an important role in silencing tumor suppressor genes, such as BRCA1, p16, and p21 in breast cancer development. It was reported that DNMT1 is over-expressed in breast cancer [29,30]. To explore the possible mechanisms of Z regulating p53 gene expression, we investigated the effects of Z on DNMT1 mRNA and protein expression in PCHNBECs and PCHBCECs after 24 h treatment. The results are shown in Figures 3 and 4. It is illustrated that Z treatment significantly increased the expression of DNMT1 mRNA and protein levels in PCHBCECs in a dose-dependent manner when compared to the control. However, Z did not significantly increase the expression of DNMT1 mRNA and protein in PCHNBECs when compared to the control. Our results suggest that Z regulate the expression of p53 in PCHBCECs, maybe through its epigenetic modification.

Breast cancer development occurs in multiple stages, which are triggered by the evolution of altered gene function. Many of the gene changes that disrupt their coding regions are involved in initiation, promotion and progression [31]. P53 is a tumor suppressor gene which plays a crucial role in regulating cell growth following exposure to various stress stimuli. It induces growth arrest or programmed cell death (apoptosis) and it also plays an important role in the control of cell cycle checkpoints. Research found that p53 is the most frequently mutated gene in human cancers. An estimated 15–60% of breast cancers contain p53 mutations [32] and loss of p53 occurs in approximately 80% of colorectal tumors [33]. Consequently, its function has been extensively studied. It was reported that tumor cells with mutant p53 protein might be associated with poor prognosis [32].

The tumor suppressor p53 can regulate both cell proliferation and apoptosis. An imbalance between cell proliferation and apoptosis results in a rapid increase in cell number, the most prominent characteristic of tumors. Normal breast epithelial cells induce p53-dependent apoptosis and p53-independent cell cycle arrest of breast cancer cells [34]. In addition, p53 was also expected to play an important role in cancer treatment with its mutation predicting a substantially worsened prognosis [33]. Several retrospective studies have suggested p53 gene mutation as an adverse prognostic indicator in breast cancer patients; it can predict early recurrence, sensitivity to chemotherapy, and death in breast cancer patients [35].

Recently, it is becoming understood that epigenetic events, or heritable changes in gene expression capacity without DNA sequence alterations, are also central to tumor progression. Understanding the differential roles of regional hypermethylation and global hypomethylation in breast cancer will facilitate novel therapeutic approaches to breast cancer therapy. It was found that hypomethylation of the global genome can lead to genomic instability that is exemplified by misalignments, DNA breakage, deletions and duplications during DNA replication [36].

DNA methylation is one of the major epigenetic modifications in cancer development which has a long-standing relationship with gene inactivity and has been implicated as an important factor in gene silencing [37,38]. In humans and most mammals, DNA methylation is the only known natural modification of DNA and only affects cytosines at the 5′ position when it is followed by a guanosine, known as CpG dinucleotides. Depending on the cell types and tissue, 3–4 % of all cytosine residues in vertebrate DNA may be present as 5-methylcytosine and approximately 70 to 80% of all CpG dinucleotides in human DNA are methylated [39]. However, this methylation occurs primarily in areas where CpG density is low, or at repeat DNA sites. Most CpG islands, especially those located in promoter region of a specific gene, are completely unmethylated. Interestingly, fully methylated CpG islands are found only in the promoters of silenced alleles for selected imprinted autosomal genes and multiple silenced genes on the inactivated X-chromosomes of females [40]. It is well established that almost half of the tumor suppressor genes that cause familial cancers through germline mutations can be inactivated in association with promoter hypermethylation in sporadic cancers [41]. Huang and colleagues, using a novel DNA array based technique called differential methylation hybridization, performed a genome wide screen for hypermethylated CpG islands in a panel of breast cancer cell lines. They found that approximately 10% of CpG islands exhibited methylation in normal breast epithelial cells. In contrast, breast cancer cell lines show methylation at a considerably larger fraction of CpG islands (15–25%) [42]. In clinical research, it was reported that DNA methylation in serum of breast cancer patients may be considered as an independent prognostic marker [43]. Methylation-dependent gene silencing is now thought to be mediated through local changes in chromatin conformation that limit promoter accessibility [44].

DNA methyltransferases (DNMTs) are critical enzymes that regulate DNA methylation. DNMT1 is the most important one; the N-terminus of DNMT1 contains regions responsible for targeting the enzyme to replication foci [45], as well as for discriminating between unmethylated and hemi-methylated DNA [46]. Overexpression of all the DNMTs at the mRNA level has been shown for several cancers [47–49]. Conversely, a mouse model for colon cancer demonstrated that reducing DNMT1 expression and activity via hemizygous knockout of the gene and treatment with a DNMT1 inhibitor greatly reduced the number of intestinal adenomas [50]. Similarly, Macleod and Szyf found that transfection of a murine adrenocortical tumor cell line with DNMT1 antisense expression vectors resulted in general DNA hypomethylation, growth inhibition in vitro, and decreased tumorigenicity in vivo [51]. Our laboratory recently reported PTP γ expression was reduced in breast cancer cell lines, SK-Br-3 and MCF-7 cells, and it can be re-activated with the treatment of deoxy-5-azacytidine, a well-known DNMT1 inhibitor [52]. These results suggest that DNMT1 can be thought of as a new target for novel drug development.

Our data indicated that 24 h Z treatment stimulated both PCHNBEC’s and PCHBCEC’s proliferation in a dose-dependent manner, and PCHBCECs were more sensitive to Z than PCHNBECs. Z increased the DNMT1 expression in PCHBCECs and decreased the p53 expression at both the mRNA and protein levels; however, no such effect was found in PCHNBECs, suggesting Z might be more harmful to cancer patients than normal and Z might have adverse health problem when cancer patients intake beef products produced by Z-implanted beef cattle containing Z or its metabolites. One of the mechanisms is due to the increase of DNMT1 and decrease of p53 expression. In addition, all our experimental data was collected from matched human breast tissues, which can provide better data for analyzing the difference between PCHNBECs and PCHBCECs with the same treatment of different concentrations of Z to decrease the possible influence of age, sex, medication, life style, etc.

In summary, our data show PCHBCECs may be more sensitive to exposure to Z. The stimulatory effects of Z on PCHBCECs might be possible through down-regulation of the expression of the p53 gene and up-regulation of DNMT1 mRNA and protein. This result suggests that suppressed expression of the p53 gene in PCHBCECs might be mediated through its epigenetic modification. Further investigation into this critical issue is in progress in our laboratory.

Human normal and cancerous breast tissues from the same patient were obtained through the Tissue Procurement Program of The Ohio State University Comprehensive Cancer Center Hospital in Columbus, OH, USA. After tissues were transferred in our laboratory, they were minced and then digested using digestion buffer which consisted of phenol red-free high calcium Dulbecco’s modified Eagle’s medium and Ham’s F12 medium (1:1) (DMEM/F12) (1.05 mM CaCl₂) with 2% Bovine Serum Albumin (BSA) (Invitrogen, Carlsbad, CA, USA) containing 10 ng/mL Cholera toxin (Sigma, St. Louis, MO, USA), 6300 U/mL Collagenase (Invitrogen), and 100 U/mL Hyalurinidase (Calbiochem, Gibbstown, NJ, USA). After the mixture was incubated in a humidified incubator (5% CO₂, 95% air, 37 °C) overnight, the solution was transferred to a 50 mL tube and centrifuged at 1200 rpm for 5 min. The upper layer containing pre-adipocytes and middle layer containing stromal cells were transferred to another 15 mL tube separately while the pellet containing epithelial cells remained in the tube. All the pellets were washed by DMEM/F12 medium with antibiotic-antimycotic (100 U/mL penicillin G sodium, 100 μg/mL streptomycin sulfate and 0.25 μg/mL amphotericin B) (Invitrogen) and centrifuged again. This wash procedure was repeated three times. The final pellet in the tube contains PCHBCECs or PCHNBECs. The pellet was then resuspended in 10 mL low calcium (0.04 mM CaCl₂) DMEM/F12 medium supplemented with 10% of low calcium FBS (Atlanta Biologicals, Norcross, GA, USA) and then transferred into a T75 flask for culturing. The method and the specific cultural medium ensure the purity of PCHBCECs or PCHNBECs isolated from normal or cancerous breast tissues. Our lab members have determined the purity of the isolated primary cultured human normal and cancerous breast epithelial cells or stromal cells by using immunocytochemistry [53].

The isolated PCHNBECs and PCHBCECs were cultured in 75 cm² culture flasks in a humidified incubator (5% CO₂, 95% air, 37 °C) with 10 mL low calcium (0.04 mM CaCl₂) DMEM/F12 mixture (Atlanta Biologicals, Norcross, GA, USA) supplemented with 10% of Chelex-100 (Bio-Rad Laboratories, Richmond, CA, USA) treated FBS (Invitrogen). The low calcium DMEM/F12 medium was changed every two days [10]. This was done to ensure the purity of PCHNBECs and PCHBCECs. When the cells grew to 85–90% confluence, cells were washed with 10 mL of calcium- and magnesium-free Phosphate Buffered Saline (PBS, pH 7.3), and then trypsinized with 3 mL of 0.25% trypsin–5.3 mM EDTA (Invitrogen) for 5–8 min at 37 °C. The trypsinization was stopped by addition of 10 mL of DMEM/F12 medium with 10% FBS. After centrifugation, the dissociated cells were resuspended in low calcium DMEM/F12 medium with 10% low calcium FBS and sub-cultured into 75 cm² culture flasks at a ratio of 1 flask to 3 flasks. All experiments were conducted on primary cultured human normal breast epithelial cells not generated beyond the fourth passage.

A total volume of 100 μL medium containing 5000 PCHNBECs or PCHBCECs/well was seeded in 96-well plates in low calcium DMEM/F12 medium and incubated at 37 °C for 24 h. The following day, medium was replaced by 100 μL low calcium DMEM/F12 supplemented with 0.2% BSA and incubated at 37 °C for another 24 h. After, the treatment of 7.5, 15, 30 nM Z was given for 24 h (0.1% DMSO to control group), the proliferation of PCHNBECs and PCHBCECs was measured by adding 20 μL of a fresh mixture of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and phenazine methosulfate (PMS) (20:1) solution (Promega, Madison, WI, USA) to each well. After incubation at 37 °C for 1–3 h, optical density were measured by kinetic microplate reader (Molecular Devices Cooperation, Menio Park, CA, USA) at 490 nm wavelength and cell growth was compared.

A total of 1 × 10⁵ viable PCHNBECs and PCHBCECs/well were seeded in 6-well plates in 5 mL low calcium DMEM/F12 medium supplemented with 10% of Chelex-100 (Bio-Rad Laboratories, Hercules, CA, USA) treated FBS (Invitrogen). After 24 h, medium was replaced with low calcium DMEM/F12 supplemented with 10% dextran coated charcoal (DCC) stripped FBS, and the cells were cultured overnight. After PCHNBECs and PCHBCECs were treated with 0.1% DMSO, 7.5, 15 and 30 nM Z for 24 h, total RNA was isolated in 1 mL TRIZOL Reagent (Invitrogen) according to the manufacturer’s instructions. RNA concentration was measured by DU-70 spectrophotometer (Beckman Instruments Inc. Fullerton, CA, USA). RNA (1 μg) from cultured cells was reverse transcribed with 200 UM-MLV Reverse Transcriptase (Invitrogen) at 37 °C for 50 min then 70 °C for 15 min in the presence of 1 μL 10 mM dNTP (10 mM each dATP, dGTP, dCTP, and dTTP at neutral PH) (Invitrogen), 1 μL 50 μM Random hexamer (Amersham, Piscataway, NJ, USA), 10 μL 5× First Strand buffer, 5 μL 0.1 M DTT and 1 μL RNase Inhibitor (Invitrogen) in a total volume of 50 μL in a gradient mastercycle (Eppendorf^®, Westbury, NY, USA).

After cDNA was synthesized, real-time PCR was conducted to amplify p53, DNMT1 and 36B4 genes. The PCR reactions for p53, DNMT1 and 36B4 were performed using the SYBR Green I detection chemistry system and detected with the Stratagene M3005XP system (Agilent Technologies, Cedar Creek, TX, USA). For each PCR run, a mixture contained 10 μL 2× SYBR^® Green PCR master mix (Applied Biosystems, Foster City, CA, USA), 2 μL newly synthesized cDNA, 5 μL primer mixer and 3 μL PCR grade water in a total volume of 20 μL. The thermal cycling condition comprised an initial step at 50 °C for 2 min, 95 °C for 10 min, and 45 cycles at 95 °C for 15 s and annealing and elongation at 55 °C for 1 min. Dissociation curves were also conducted after amplification to ensure the reaction specificity. The primer sequences for p53 were 5′-GCT CCT GTG CTG CGA AGT GG-3′ (sense) and 5′-TGG AGG CGT CGG TGT AGA TG-3′ (antisense, product size 372 bp). The primer sequences for DNMT1 were 5′-CAT TTT ATC CCC ATT GAG AAG TA-3′ (sense) and 5′-CTG AAA ATT AAG TCC TTG TGC CCA G-3′ (antisense, product size 273 bp). The primer sequences for 36B4, an internal control were 5′-AAA CTG CTG CCT CAT ATC CG-3′ (sense) and 5′-TTT CAG CAA GTG GGA AGG TG-3′ (antisense, product size 563 bp). The results were presented as the ratio of p53 or DNMT1 to 36 B4.

PCHBCECs and PCHNBECs were plated in a 10 cm culture dish with a density of 1 × 10⁶ viable cells/dish within 10 mL low calcium DMEM/F12 supplement with 10% FBS and cultured overnight. The media was replaced with low calcium DMEM/F12 supplemented with 5% DCC treated FBS and was cultured for another 24 h. PCHBCECs and PCHNBECs treatment were the same as previously described. After 24 h treatment, proteins were extracted from each treatment group using M-PER^® mammalian protein extraction reagent (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Protein concentrations were measured using a Micro BCA™ protein assay reagent kit (Pierce, Rockford, IL, USA) following the manufacturer’s protocol. Proteins (50 μg) from each treatment group were separated by 4–15% Tris-HCl gel electrophoresis and then transferred to a Polyvinylidine Fluoride (PVDF) membrane (Bio-Rad Laboratory, Hercules, CA, USA). The membrane was first blocked in Phosphate Buffered Saline −0.1% Tween 20 (PBST) containing 10% fat free dry milk for 1 h and then incubated with DNMT1 goat polyclonal antibody (dilution 1:500, sc-10219, Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA), p53 antibody (1:1000 dilution, Cell Signaling Technology^® Danvers, MA, USA) and β-actin antibody (1:2000 dilution, Santa Cruz Biotechnology, Inc.) for 1 h. The membrane was rinsed in PBST three times, each time for 5 min. In the following step, the membrane was incubated with second antibody (donkey anti-goat IgG-horseradish peroxidase HRP for DNMT1 and β actin; ECLTM anti-rabbit IgG-HRP for detecting p53) for 1 h. After washing the membrane in PBST three times, p53, DNMT1, and β-actin protein were visualized with a chemiluminescent detection system (ECL, Amersham Pharmacia Biotech, Buckinghamshire, UK). Pictures were taken using a FujiFilm LAS-300 imaging system (FUJIFILM Medical Systems USA, Inc.). The protein ratios of p53 to β-actin and DNMT1 to β-actin were calculated by measuring the density of the specific band using Multi-Gauge software (v3.0).

The results for the cell proliferation assay are presented compared to the control group as mean ± standard deviation (SD) for 4 replicate culture wells. The results for the mRNA expression are presented compared to the control group as mean ± standard deviation (SD) for 3 replicates. Analyses were performed using Minitab 15 software (Minitab Inc. PA, USA). Statistical differences were determined by using two sample t-test or ANOVA analysis for independent samples. P-values of less than 0.05 were considered statistically significant differences.