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Cancer Research 67, 3698, April 15, 2007. doi: 10.1158/0008-5472.CAN-06-4000
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Synergistic Effect of Cyclin D1 and c-Myc Leads to More Aggressive and Invasive Mammary Tumors in Severe Combined Immunodeficient Mice

Yong Wang, Archana Thakur, Yuan Sun, Jack Wu, Hector Biliran, Aliccia Bollig and D. Joshua Liao

Department of Pathology, Barbara Ann Karmanos Cancer Institute, Hudson Webber Cancer Research Center, Wayne State University School of Medicine, Detroit, Michigan

Requests for reprints: D. Joshua Liao, Hormel Institute, University of Minnesota, 801 16th Avenue, NE, Austin, MN 55912. Phone: 507-437-9665; Fax: 507-437-9606; E-mail: djliao{at}hi.umn.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cyclin D1 is one of the most commonly overexpressed oncogenes in breast cancer; yet, it is not clear whether cyclin D1 alone is capable of causing malignant transformation of mammary epithelial cells. Here, we show that ectopic expression of cyclin D1 in benign mouse mammary epithelial cells promotes cell proliferation, anchorage-independent growth in soft agar, and tumorigenesis in severe combined immunodeficient mice. To address the possible interaction of cyclin D1 and c-myc in malignant transformation, we used cyclin D1/c-myc dual-expressing clones, which displayed more aggressive and invasive phenotype than cyclin D1–expressing clones. These data provide evidence that overexpression of cyclin D1 or coexpression with c-myc could cause invasive malignant transformation of benign mouse mammary epithelial cells. Furthermore, microarray analysis of cyclin D1 and cyclin D1/c-myc clones showed that these two tumor-producing clones might use distinct invasive pathways. In summary, overexpression of cyclin D1 may commit mammary epithelia to a tumor-prone phenotype in which cooperation with other genes, such as synergy with c-myc, may lead to a more aggressive phenotype. [Cancer Res 2007;67(8):3698–707]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The cyclin D1 oncogene is overexpressed or amplified at high frequencies in a variety of human cancers. In human breast cancer, such alterations are found in ~50% of cases (1). Targeted deletion of the cyclin D1 gene has shown its essential role in normal mammary gland development (2–4). Overexpression of cyclin D1 in the mammary gland of the transgenic mice driven by mouse mammary tumor virus (MMTV) long terminal repeat causes hyperplasia, but adenocarcinomas develops after a long latency (18 months) and at a low penetrance (5). Animal studies using knockout, knock-in, and combined knockout with transgenic approaches suggest that mammary carcinogenesis initiated by several oncogenes, such as neu and Ha-ras, are solely dependent on cyclin D1 (4–6), which further strengthens the importance of cyclin D1 in breast cancer formation.

Studies using in vitro culture systems to test the oncogenic potential of cyclin D1 were largely uninformative and showed conflicting data reporting three different conclusions in both fibroblastic and epithelial cell systems: (a) that overexpression of cyclin D1 alone only transforms fibroblasts, such as Rat6 and NIH3T3, that can form tumors in severe combined immunodeficient (SCID) mice (7, 8); (b) that overexpression of cyclin D1 in Rat6 and NIH3T3 fibroblasts can promote cell proliferation, but cyclin D1 alone is not capable of promoting tumorigenesis (9); (c) that cyclin D1 inhibits cell proliferation and induces apoptosis in mouse mammary epithelial cells (10). Clearly, these conflicting data raise an issue whether cyclin D1 alone is capable of causing malignant transformation of cells, especially mammary epithelial cells.

Like cyclin D1, the c-myc oncogene has also been shown to be aberrantly expressed or amplified in most, if not all, types of human malignancy, including breast cancer, suggesting that the c-Myc oncoprotein plays an important role in cancer development and progression (11). MMTV-c-myc transgenic mice develop mammary tumors at 9 to 12 months of age and, basically, at 100% tumor penetrance (12). Unlike mammary carcinogenesis induced by neu and H-ras, mammary carcinogenesis induced by MMTV-c-myc or Wnt does not require cyclin D1 because tumors can develop in cyclin D1 knockout mice (4). This is not surprising as c-myc has been shown in several in vitro systems to inhibit cyclin D1 expression (13–17) and attenuate the cell cycle–dependent cyclin D1 oscillation (18). Moreover, c-Myc–modulated cell cycle progression does not require cyclin D1 (19) but, instead, can be elicited via induction of cyclin D2 in at least certain cell types (20–23). Collectively, these data indicate a possibility that c-Myc and cyclin D1 may mediate mammary carcinogenesis via different pathways. Now the question is whether the c-Myc–mediated and cyclin D1–mediated pathways have any interaction that can result in additive or synergistic effects on tumor progression, such as cell proliferation, invasive ability, and metastasis potential, if both pathways are activated simultaneously.

In this study, we used a benign mouse mammary epithelial cell line (NMuMG) to ectopically overexpress cyclin D1 alone or with c-myc and subsequently tested these cell clones for cell proliferation, anchorage-independent growth in soft agar, and tumorigenesis in SCID mice. Results show that cyclin D1 either alone or in collaboration with c-myc can cause malignant transformation of mouse mammary epithelial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and plasmids. The mouse mammary epithelial cell line NMuMG obtained from the American Type Culture Collection (Manassas, VA) was used to generate the stably transfected cell clones. NMuMG is generally described as normal mammary epithelial cells in the literature, as it is abbreviated, but we rather consider it a benign cell line. NMuMG cells were cultured in DMEM (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS; Invitrogen) at 37°C and 5% CO2. The pcDNA3.1/Neo-CCND1 or pcDNA3.1/Hygro-myc plasmids were used to stably transfect the NMuMG cells to overexpress the murine cyclin D1 and c-myc genes, respectively. The cyclin D1 (Genbank accession no. NM_007631) and c-myc (Genbank accession no. NM_010849) inserts encode the entire cDNA sequence, in addition to franking 5' untranslated sequence and part of 3' sequence beyond the stop codon. The constructed plasmids were verified by DNA sequencing. The empty vector was used to generate stable control vector clones.

Single and double transfection. Transfection was done using LipofectAMINE 2000 (Invitrogen) according to manufacturer's instructions. Briefly, parental NMuMG cells were cultured in 24-well plates at 80% to 90% confluency. Plasmid DNA (1.0 µg per well) was fully mixed with 10 µL LipofectAMINE 2000; this mixture was then used to transfect the NMuMG cells. After 24 h of treatment, cells were trypsinized and diluted (1:10 to 1:15) to be replated into 10-cm culture dishes. Culture medium with selection drug (800 µg/mL neomycin or 400 µg/mL hygromycin) was changed every 3 to 4 days. Single cell clones were isolated for clone expansion. Each cell clone was screened by reverse transcription-PCR (RT-PCR) and Western blot to determine the exogenous cyclin D1 expression at both mRNA and protein levels. The transfected cell clones were maintained and passaged in culture medium with neomycin (400 µg/mL) or hygromycin (200 µg/mL). After 3 months (about 25 passages), the stably transfected cell clones were used for studies.

Double-stable clones were generated without the lead time, just after four to five passages of cyclin D1 or c-myc stable clones. For double transfection, the pcDNA3.1/Hygro-myc or pcDNA3.1/Neo-CCND1 plasmids were used to transfect the stable clones of cyclin D1 or c-myc, respectively. Similar to single-transfected clones, the double-transfected cell clones were maintained and passaged in culture medium with neomycin (400 µg/mL) and hygromycin (200 µg/mL) and used after 20 to 25 passages for in vitro or in vivo experiments.

RNA extraction, RT-PCR, and genotyping. Total RNA was extracted from transfected cells using Trizol reagent (Invitrogen) according to the manufacturer's instruction and stored at –80°C. RT-PCR was used to identify the stable cell clones transfected with cyclin D1 and c-myc using Taqman RT kit (Applied Biosystems, Foster City, CA) and Taq Ready Mixture kit (Sigma, St. Louis, MO). The specific primer pairs for identification of constructed cyclin D1 include an upstream primer located in CCND1 insert (cyclin D1 exon 4, 5'-CTACCGCACAACGCACTTTC-3') and the downstream primer located in vector (BGH, 5'-TAGAAGGCACAGTCGAGG-3'), which amplified a special RT-PCR product that can only originate from the cyclin D1 expression vector in transfected clones. Similarly, the specific primer pairs were used to identify the mRNA expression of exogenous c-Myc (c-Myc-1F, 5'-CACCGCCTACATCCTGTCCATTCAAGC-3' and BGH, 5'-TAGAAGGCACAGTCGAGG-3'). The vector control clones were identified by using T7 and BGH primer pairs (T7, 5'-TAATAC-GACTCACTATAGGG-3'; BGH, 5'-TAGAAGGCACAGTCGAGG-3'). Competitive RT-PCR was done to examine the mRNA expression of exogenous and endogenous mRNA expression of cyclin D1, using the same upstream primer (cyclin D1 exon 4, 5'-CTACCGCACAACGCACTTTC-3'), but different downstream primers specific for the endogenous cyclin D1 (D1-E5 stop-r, 5'-TCAGATGTCCACATCTCGCACG-3') or for the exogenous cyclin D1 (BGH, 5'-TAGAAGGCACAGTCGAGG-3'). Multiple primer RT-PCR was employed to examine the mRNA expression of exogenous cyclin D1 and c-myc in double stable cell clones, using upstream primers (D1 STOP-F2, 5'-GTGCAGGATGTGGACATCTG-3' and c-Myc-1F, 5'-CACCGCCTACATCCTGTCCATTCAAGC-3') and the same downstream primer (BGH, 5'-TAGAAGGCACAGTCGAGG-3'). Genotyping by RT-PCR was also done to test the exogenous gene expression in tumors formed from stable cell clones in SCID mice using above primers. Other primers used in semiquantitative RT-PCR and quantitative RT-PCR are listed in Table 1 .


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  		Table 1. Primer sets for semiquantitative RT-PCR

 
Western blot. Methods for the preparation of protein samples and immunoblotting were described previously (24). The total protein in cell lysate was quantitated using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Protein aliquots (25 µg per lane) were electrofractionated on SDS-PAGE and transferred to nitrocellulose membrane for detection by Western blot using rabbit polyclonal anti-cyclin D1 (sc-717; Santa Cruz Biotechnology, Santa Cruz, CA) or anti-c-Myc antibody (sc-20; Santa Cruz Biotechnology), and proteins were visualized by chemiluminescence (Pierce).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cells were seeded in 96-well plate at 1500 cells per 100 µL per well concentration. Cells were grown in culture medium containing either 1.0%, 2.5%, 5.0%, and 10% FBS or 1, 10, and 100 ng/mL epidermal growth factor (EGF) or insulin for 96 h. At the end of incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (40 µL per well of 5 mg/mL MTT in PBS) was added to each well, and plates were incubated in the dark for 3 h at 37°C. After removal of the medium, the dye crystals were dissolved in isopropanol, and viable cells were detected by reading the absorption at 595 nm in the Ultra plate reader (Tecan Research, Triangle Park, NC). Experiments were repeated thrice in quadruplicate wells to ensure the reproducibility of results.

Soft agar growth assay. Anchorage-independent growth was assessed by colony formation in soft agar growth assay. Briefly, equal volumes of agar (1%, DNA grade) and 2x DMEM (with 20% FBS) were mixed at 40°C to make 0.5% agar in six-well tissue culture plates (Corning, Corning, NY) as a base agar. Cells (0.1 mL, 2.0 x 105 cells per mL) were suspended in 3 mL of 2 x DMEM (with 20% FBS) and 3 mL of 0.7% agar; 1.5 mL cell suspension was then added in each well (as 0.35% top agar) with final concentration of 5,000 cells per well. Top agar was covered with culture medium. Plates were incubated at 37°C and 5% CO2 in humidified incubator for 18 days, and medium was changed every 3 to 5 days. Colony formation was observed by light phase contrast and also stained with 0.5 mL of 0.01% Crystal Violet in PBS for 45 min at room temperature followed by photomicrography. Experiments were repeated twice to ensure the reproducibility of our results.

Tumorigenic activity in SCID mouse and histologic examination. Female SCID mice (Taconic, NY) were housed in sterile condition for 1 week before the experiments. At ~7 weeks of age, cells (107 cells in 0.2 mL PBS) of various NMuMG derived clones were injected into the second/third pair of mammary fat pad of both left and right sides. The number of animals for each cell clone was indicated in details in results. Mice were monitored every alternative day for the development of palpable xenograft tumors. The animals were sacrificed when the xenograft tumors reached the ethical limit (about 15–20 mm in diameter), or when the study was terminated at 19 weeks after injection. In a second set of animal study, we injected cells (107 cells) in to the peritoneal cavity of female SCID mice at the age of 7 weeks and sacrificed the mice 8 weeks later. In a third set of animal experiments, cells (107 cells) were injected into the left cardiac ventricle of female SCID mice at 9 weeks of age, using the injection procedure described in details by Yin et al. (16). After scarification, the animals were examined for metastasis in the lung, liver, kidney, and brain, and metastatic tumors were fixed in 10% formalin for further histologic examination. All animal experiments were carried out by strict abidance to the protocols approved by the Animal Investigation Committee of Wayne State University.

Microarray experiments and data analyses. Total cellular RNA was prepared using RNeasy mini kit (Qiagen, Valencia, CA) from two stable clones from each condition (cyclin D1–overexpressing ND5 and ND11, double cyclin D1/c-myc–overexpressing clones-ND5/C23 and ND11/C5, and vector control clones NN2 and NN2/NH2). Assurance of quality assessment and microarray analysis were carried out by personnel in the Applied Genomics Technology Center (Center of Molecular Medicine and Genetics, Wayne State University) and repeated at least twice to ensure the reproducibility and to draw the statistically significant conclusions. Approximately 5 µg of RNA was used to obtain the cRNA according to standard Affymetrix protocols. The cRNAs were hybridized with the High Density Mouse Genome M430-2 containing 45101 probesets (Affymetrix, Santa Clara, CA). Microarray analysis was done as described previously (25). Data analysis was done with the GeneSifter and GeneSpring software version 7.2. Expression was normalized against the Affymetrix spike RNA levels as well as against glyceraldehyde phosphate dehydrogenase and actin mRNA levels in all the samples. The expression of genes in cyclin D1 or double-transfected clones with cyclin D1 and c-Myc were compared with the vector control clones as the baseline. Selected candidate genes were confirmed by semiquantitative RT-PCR analysis.

Matrigel invasion assay. The cyclin D1, cyclin D1/c-myc–overexpressing, or vector control cells were suspended at 1 x 106 cells per mL in medium containing DMEM with 0.1% bovine serum albumin, and 100 µL cell suspension was added to the upper well of Transwell inserts coated with 1 mg/mL Matrigel (BD Biosciences, San Jose, CA). In lower wells, 0.6 mL medium containing CXCL12 (10 nmol/L) or vehicle was added. The plates were incubated for 24 h at 37°C in 5% CO2. After incubation, the inserts were carefully lifted; cells from the upper surface were gently removed; and the remaining cells at the bottom side of the filter were fixed and stained using the Diff-Quick Staining. The results are expressed as the percentage of cells that invaded the Matrigel and migrated to the bottom side of the Transwell membrane relative to the vector control cells. Each experiment was repeated at least thrice in triplicates to ensure the reproducibility of our results.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of stable cyclin D1, c-myc, or double cyclin D1/c-Myc clones. Stable clones of NMuMG cells overexpressing cyclin D1 were generated by transfection with a pcDNA3.1/neo-CCND1 plasmid. NMuMG cells were also transfected with neo-pcDNA3.1 empty vector to establish control cell clones. Results of competitive RT-PCR showed two specific products representing the total mRNA (endogenous + exogenous) and exogenous cyclin D1 in several stable cyclin D1 cell clones, whereas only one specific product representing endogenous cyclin D1 mRNA was seen in the vector control clones. Figure 1A (top) shows the expression of cyclin D1 mRNA and protein in representative cyclin D1 clones ND5 and ND11 as well as vector control clone NN2. Similarly, NMuMG cells were transfected with the pcDNA3.1/Hygro-myc plasmid or empty vector to generate several stable c-myc cell clones and control cell clones. The expression of exogenous c-myc mRNA and protein were shown in Fig. 1A (middle). Typically, cyclin D1 clone cells showed about 4- to 5-fold higher expression of cyclin D1, whereas c-myc clone cells showed about 5- to 7-fold higher expression levels of c-myc, compared with the corresponding empty vector–expressing cells. Expression levels remained unchanged after 25 passages, suggesting that exogenous cyclin D1 and c-myc were stably overexpressed.


Figure 1
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  		Figure 1. A, generation and identification cyclin D1–, c-Myc–, cyclin D1/c-Myc–, or c-Myc/cyclin D1–expressing stable clones. Top, exogenous and total (endogenous + exogenous) cyclin D1 mRNA expression in stable cell clones transfected with either cyclin D1 construct (ND5 and ND11) or empty vector (NN2) by RT-PCR and a specific cyclin D1 protein band at 36 kDa by Western blot analysis. ND5 and ND11 show ~4- to 5-fold higher cyclin D1 protein levels compared with vector control clone-NN2. Middle, exogenous and total c-Myc mRNA expression in stable cell clones transfected with either c-Myc construct (NC2 and NC3) or empty vector (NH2) by RT-PCR and a specific c-Myc protein band at 64 kDa by Western blot. NC2 and NC3 show ~5- to 7-fold higher c-Myc protein levels compared with vector control clone NH2. Bottom, double (cyclin D1 and c-Myc) transfected stable clones (ND5/C23, ND11/C5, or NC2/D1, NC3/D1) showing two bands, a specific cyclin D1 protein band at 36 kDa, and c-Myc protein at 64 kDa by Western blot analysis. B, morphologic changes in cyclin D1–transfected NMuMG cells compared with empty vector–transfected cells. C, top, cyclin D1 (ND5)– and cyclin D1/c-Myc (ND5/c-Myc)–transfected stable clones show significantly increased (P < 0.02) cell proliferation with 10%, 5%, or 2.5% FBS compared with vector control (NN2 and NH2), c-Myc (NC2)–, or c-Myc/cyclin D1 (NC2/D1)–expressing clones but not with lower than 2.5% FBS. Middle and bottom, EGF and insulin (1, 10, and 100 ng/mL and 10% FBS) did not collaborate with overexpressed cyclin D1 or c-Myc to enhance cell proliferation. Experiments were repeated thrice. Columns, mean from representative experiment; bars, SEM. D, cyclin D1–transfected (ND5 and ND11) or double-transfected (ND5/C23 and ND11/C5) stable clones show the ppRb protein only; vector control clone (NN2) shows both pRb and ppRb proteins.

 
We further transfected the ND5 and ND11 cells with a pcDNA3.1/Hygro-myc plasmid and selected clones that expressed both cyclin D1 and c-myc. In addition, double-transfected cells were also generated by transfection of c-myc–expressing stable clones with the pcDNA3.1/neo-CCND1 plasmid. The representative clones overexpressing cyclin D1 and c-myc (named ND5/C23, ND11/C2, and ND11/C5) or c-myc–expressing clones transfected with cyclin D1 (named NC2/D1 and NC3/D1) were used in further studies. Figure 1A (bottom) shows the Western blot detection of cyclin D1 and c-Myc proteins in these clones. We also established several stable clones of NMuMG cells expressing both pcDNA3.1neo and pcDNA3.1hygro empty vectors as controls, such as NN2/NH2 clone.

Overexpression of cyclin D1 alone changes cell morphology and increases cell growth. We observed that cyclin D1–expressing cells showed changed morphology several days after transfection before the first passage, compared with the empty vector–transfected cells (Fig. 1B). We used MTT assay to assess the cell proliferation of the cyclin D1–, c-myc–, or double-transfected stable clones by measuring the number of viable cells at different time points. Results showed that overexpression of cyclin D1 alone promoted cell proliferation in the presence of 10%, 5%, and 2.5% serum conditions in a dose-dependent manner compared with the neo-vector control clones, data from representative clones are shown in Fig. 1C (top). Addition of 1, 10, or 100 ng/mL EGF (Fig. 1C, middle) or insulin (Fig. 1C, bottom) failed to promote the proliferation of cyclin D1– or double cyclin D1 and c-myc–transfected cells under low serum (1% FBS) concentration. These data suggest that cyclin D1 is capable of driving proliferation of mouse mammary epithelial cells only at serum concentrations ≥2.5% indicating that certain serum-derived factors, excluding EGF and insulin, are required to cooperate with cyclin D1 to promote proliferation and tumorigenesis. Similar to cyclin D1 clones, the double cyclin D1 and c-myc–transfected cells also showed increased proliferation compared with vector control (NN2 or NH2) clones (Fig. 1C, top). On the contrary, c-myc– or c-myc/cyclin D1–overexpressing clones did not show increased number of viable cells compared with vector control clones under 10%, 5%, or 2.5% serum (Fig. 1C, top). This could be due to increased cell death in these clones compared with cyclin D1 or cyclin D1/c-myc clones because we noticed more floating cells in the wells seeded with the c-myc and c-myc/cyclin D1 clones during our routine microscopic visualization. Furthermore, we determined the phosphorylation status of retinoblastoma protein (pRb) in cyclin D1– or cyclin D1/c-myc–overexpressing clones; results showed the presence of hyperphosphorylated Rb (ppRb) only in these clones, whereas vector control cells showed both pRb and ppRb (Fig. 1D).

Overexpression of cyclin D1 leads to malignant transformation of benign mouse mammary epithelial cells. To examine whether overexpression of cyclin D1 alone can cause malignant transformation, an anchorage-independent growth assay in soft agar was employed. A large number of colonies were formed in cyclin D1–overexpressing cells at 18 days after cell seeding, whereas the vector control cells failed to develop colonies. Representative data are presented in Fig. 2A and B . Interestingly, c-myc cell clones (NC2 and NC3) did not develop obvious colonies at the same time point. Other cyclin D1 clones but not c-myc clones also showed anchorage-independent growth correlating to cyclin D1 levels (data not shown).


Figure 2
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  		Figure 2. Tumorigenicity of cyclin D1, cyclin D1/c-Myc, and vector control clones. A, an anchorage-independent soft agar growth by staining with crystal violet. Stable cell clones transfected with cyclin D1 or cyclin D1/c-Myc (ND5 and ND5/C23) displayed an increased anchorage-independent growth in soft agar compared with neo control cell clone-NN2. c-Myc alone–transfected clones did not show any growth in soft agar; however, further transfection of these clones with cyclin D1 resulted in slight growth in soft agar. B, soft agar growth by light phase-contrast micrography (at x10 and x50 magnification) of ND5- and ND5/C23–seeded plates. C, tumorigenicity of cyclin D1 and cyclin D1/c-myc stable cells in SCID mice. a, ND5/C23 cells inoculated in the mammary fat pad developed a tumor that was palpable at 2 wks and reached the ethical limit 9 wks after injection. b, NN2 cells develop a small palpable lump in the mammary fat pad 19 wks after injection, but histologic examination confirmed it as a pseudo-tumor consisting of proliferating ductal cells and stroma. c, typical histology of ND5 cell–derived malignant xenograft tumor. d, typical histology of ND5/C23 cell–derived malignant xenograft tumors. e, a lung metastatic tumor developed from the ND5/C23 cells injected in the heart. f, chest tumor seeds of ND5/C23 cells injected into the chest cavity. D, genotyping of tumor samples from the cyclin D1 (ND5)– or cyclin D1/c-myc (ND5/C23)–injected clones in SCID mice showing that tumors maintained the overexpression of exogenous cyclin D1 and/or c-myc.

 
We then injected cells from ND5 cells into mammary fat pads of five female SCID mice; all mice developed palpable xenograft tumors at both left and right injection sites between 10 and 12 weeks. At 19 weeks when animals were euthanized, the palpable tumors grew about 10 to 12 mm. To confirm these results, in a second set of animal study, we injected cells from three cyclin D1 clones (ND2, ND5, and ND11) to SCID mice (n = 5 mice per clone; two sites). All mice developed palpable tumors between 10 and 13 weeks and reached the ethical limit between 18 and 20 weeks. The tumors showed malignant morphology (Fig. 2C, c), with some areas of invasion to the surrounding stroma. PCR analyses of these tumor samples showed that the tumors still maintained the high expression of exogenous cyclin D1 (data not shown). Only one of five mice developed a palpable lump (pseudo-tumor, 5 mm in size) at 19 weeks after injection in mice injected with vector control (NN2) cells (Fig. 2C, b; Table 2A ).


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  		Table 2.

 
To determine the metastatic potential of the cyclin D1–expressing cells, we also injected ND2, ND5, and ND11 cells into the peritoneal cavity as well as into the left cardiac ventricle (n = 5 mice per clone). Neither tumor seeds were found in the peritoneal cavity nor the macroscopic or microscopic metastasis was discerned in the lung, liver, kidney, or brain after 2 months after injection (Table 2B).

We also injected two c-myc–expressing cell clones (NC2 and NC3) into mammary fat pads of SCID mice (five animals per cell clone). Only one of five mice injected with NC2 or NC3 developed very small tumors. Results are summarized in Table 3A . Five mice were also injected with NH2 vector–expressing cells, and none of these mice developed palpable tumors 19 weeks after injection.


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  		Table 3.

 
Overexpression of cyclin D1/c-Myc leads to more aggressive in vitro and in vivo features. Next, we determined whether ectopic expression of c-myc in cyclin D1–overexpressing clones could result in additive or synergistic effects leading to enhanced anchorage-independent growth. Consistent with this notion, our results showed increased colony number and larger colony size of ND5/C23 and ND11/C5 double-expression clones in soft agar compared with the cyclin D1 alone expressing clone. The representative results are shown in Fig. 2A and B. On the other hand, the c-myc–overexpressing clones followed by transfection with cyclin D1 (NC2/D1 or NC3/D1) only showed slightly increased anchorage independent growth in soft agar compared with the c-myc–overexpressing alone clones (Fig. 2A).

To access the tumorigeneity of cyclin D1/c-myc cells, we initially injected ND5/C23 cells into the mammary fat pads of five female SCID mice; all mice developed palpable xenograft tumors at both injection sites within 12 to 15 days (Fig. 2C, a). Because tumors reached the ethical limits, mice were sacrificed between 22 and 28 days after injection. To confirm this result, in a second set of animal study, we injected ND5/C23, ND11/C2, and ND11/C5 cells in SCID mice (n = 5 mice per clone; two sites). All animals developed palpable tumors at 13 to 21 days, and the tumors reached the ethical limit (about 15–20 mm in diameter) between 21 and 34 days. These tumors manifested more aggressive features with highly invasive ability, characterized by a penetrating growth in stromal tissue, chest muscle and adhered to the chest bones; a representative photomicrograph is shown in Fig. 2C, d. PCR analyses of these tumor samples showed that they maintained the overexpression of exogenous cyclin D1 and c-myc. We also inject NC2/D1 and NC3/D1 cells (n = 5 mice per clone; two sites); none of the animals developed any xenograft tumors within 3 months (Table 2A).

To determine the metastatic potential of the double oncogene–expressing cells, we injected ND5/C23 and ND11/C5 cells (107 cells per animal) into the peritoneal cavity of SCID mice (n = 5 mice per clone). All animals exhibited many tumor seeds in the peritoneal cavity and abdominal wall with size ranging from 1 to 6 mm when sacrificed 2 months after injection. We then injected ND5/C23 and ND11/C5 cells into the left cardiac ventricle of SCID mice (n = 5 mice per clone) to further determine their metastatic ability. Because some animals showed weak health 9 weeks after injections, we terminated the study at this time point. Seven mice were found to have three to five spots of metastatic tumor spots on the lung surface, varying between 1 and 3 mm in diameter. Histologic examination confirmed the existence of multiple metastatic tumor foci/nodules in the lung (Fig. 2C, f; Table 2B). No lung metastasis was found in the remaining three mice. But in one of the mice, a large chest tumor and many tumor seeds were found on the chest walls and diaphragm (Fig. 2C, e), indicating that the cells were misrouted into the chest cavity of this mouse instead of the left cardiac ventricle.

We also injected NN2/NH2 cell clone that expressing both neo- and hygro-pcDNA3.1 empty vectors into mammary fat pads of five SCID mice (two injection sites per animal). Only one of five mice developed palpable pseudo-tumor (5 mm) when sacrificed 14 weeks after injection, with histology similar to the pseudo-tumor from NN2 cells described above (Fig. 2C, b).

Gene expression profile in single- or double-transfected cell clones. To identify the molecular pathways mediating the effect of cyclin D1 and cyclin D1/c-myc, we used cDNA microarray to determine the expression profile of two cyclin D1–overexpressing clones (ND5 and ND11) and two double-transfected (ND5/C23 and ND11/C5) clones as well as their corresponding control clones. Figure 3A shows the proportion of genes that are similar or different in cyclin D1 and cyclin D1/c-myc clones. The signal values were calculated from the multiple probes present on each chip as described earlier (25), and each chip was repeated twice to draw a statistically significant data.


Figure 3
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  		Figure 3. Gene expression pattern in cyclin D1 and cyclin D1/c-myc stable cells. A, proportion of genes that are similar or different in cyclin D1 and cyclin D1/c-myc clones. Average fold differences from representative clones (ND5 and ND5/C23) are relative to their vector controls. B, verification of some genes likely to be involved in invasion and metastasis showed expression pattern similar to microarray data upon confirmation by RT-PCR. C, fold change in mRNA expression of invasion and metastasis-related genes in cyclin D1 and cyclin D1/c-Myc cell clones by microarray analysis. D, chemoinvasive activity of cyclin D1 and cyclin D1/c-Myc clones. Photomicrographs of cells accumulated at the bottom side of the Transwell membrane at x10 and lower at x50 magnification of representative cyclin D1, cyclin D1/c-Myc, and vector control clones. Chart showing a significantly increased (P < 0.001) chemoinvasion against CXCL12 (SDF-1{alpha}) in cyclin D1 (ND5)–overexpressing clone compared with representative cyclin D1/c-Myc double-transfected (ND5/C23) or vector control (NN2) clones.

 
Selected expressed genes (>10-fold higher or >10-fold lower gene expression levels) were categorized based on their functional properties and are presented in Table 3A. We selected candidate genes from microarray data for further verification of their expression by RT-PCR (Fig. 3B; Table 3B). This selection was based on results in the literature indicating a direct or indirect role for each candidate gene in invasion/migration, proliferation, or signal transduction. The most strikingly differential expression between cyclin D1 and cyclin D1/c-myc clones was observed in genes related to invasion and migration (Fig. 3B and C). The cyclin D1–expressing cells showed up-regulated CXCL12, CXCR4, MMP-2, and MMP-12, whereas the double-expressing cells exhibited up-regulation of Snail homologue 2 (Snail2 or Slug) and significant down-regulation or complete loss of E-, N-, K-, L-, and P-cadherin and epithelial tight junction protein claudin 2 (Cldn2). Collectively, these results suggest that cyclin D1 and co-overexpressed cyclin D1/c-myc may use distinct pathways for the invasion (as observed in our xenograft model).

We further validated the microarray and RT-PCR data and tested the functionality of some of the genes related to invasion and migration. Cyclin D1– and cyclin D1/c-myc–overexpressing and vector control clones were subjected to Matrigel chemoinvasion assay to check the possible differences between these two conditions. Cyclin D1 clones (ND5 and ND11), which expressed CXCR4, showed significantly increased (P < 0.001) chemoinvasion against CXCL12 compared with either double cyclin D1/c-myc (which did not express CXCR4) or vector control clones (Fig. 3D). Although cyclin D1/c-myc cells showed increased chemokinesis, they could not pass through the pores of Transwell membrane. Figure 3D (middle) show that cyclin D1/c-myc cells are stuck in pores rather than passing through it, unlike cyclin D1–overexpressing cells that pass through the pores to the bottom side of the filter.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Literature on ectopic overexpression of cyclin D1 shows conflicting data. Cyclin D1–overexpressing rat fibroblasts are reported to show accelerated G1-S phase transition in a growth factor–dependent manner without obvious signs of transformation (9, 26), whereas Pagano et al. (27) reported that cyclin D1 overexpression prevented normal fibroblasts from entering S phase. Constitutive expression of cyclin D1 has also been shown to sensitize cells to radiation, serum starvation, and retinoic acid–induced cell death (27–29). Now in this study, we add one more conflict in the literature by showing, for the first time, clear in vitro and in vivo evidence that overexpression of wild-type cyclin D1 can drive cell proliferation and cause malignant transformation of benign mouse mammary epithelial cells, which leads to invasive tumor growth in SCID mice. It is noteworthy that the proliferative potential of cyclin D1–overexpressing stable clones depends on serum concentrations ≥2.5%, indicating that some serum factors, but not insulin and EGF, are needed to assist cyclin D1. With regard to the tumorigenic and invasive phenotypes of cyclin D1–overexpressing cells, we cannot rule out the possibility that during generation of stable cell clones, additional genetic changes may have accumulated and are likely to influence the tumorigenic or invasive phenotypes of cyclin D1–overexpressing clones. We observed that cyclin D1– and cyclin D1 plus c-myc–overexpressing clones showed increased phosphorylation of pRb. Thus, deregulation of Rb phosphorylation status may be one of the mechanisms for cell growth and malignant transformation in cyclin D1–overexpressing cells.

In our system, c-myc–overexpressing clones hardly formed colonies (only few colonies) in soft agar at the time when cyclin D1 cells already formed a number of large colonies. Consistent with these in vitro data, only 2 of 10 SCID mice injected with c-myc–expressing cells developed very small xenograft tumors with long latency, in strong contrast to the 100% xenograft tumor penetrance from cyclin D1–expressing cell clones. We concluded that cyclin D1 is more potent than c-myc in malignant transformation of benign mouse mammary epithelial cells.

Another finding of our study is that c-myc collaborates with cyclin D1 in driving cell growth to form more aggressive xenograft tumors in SCID mice. Interestingly, this only occurs in cyclin D1 stable clones transfected with c-myc (i.e., cyclin D1/c-myc cells) not in the c-myc stable clones transfected with cyclin D1 (i.e., c-myc/cyclin D1 cells). The c-myc/cyclin D1 cells show only slight growth in soft agar and do not form tumors in SCID mice, at least not as early as the cyclin D1/c-myc cells or until the termination of experiment. These results suggest that the sequence of hit may be an important determinant for deciding the cell fate, and that initial insult or existing cellular milieu plays a prominent role in determining the transforming potential of a cell. These results provide very important insights to explain some of the conflicting results seen in experiments with cyclin D1 overexpression and suggest that the cellular milieu may play a critical role.

The microarray analysis of cyclin D1– and cyclin D1/c-myc–overexpressing cell clones revealed a high degree of unique differential expression of invasion and metastatic-related genes. The cyclin D1–transfected clones showed up-regulation of CXCL12, CXCR4, MMP-2, and MMP-12, whereas the cyclin D1/c-myc–expressing clones showed up-regulation of Snail2 and significant down-regulation to complete loss of E-, N-, K-, L-, and P-cadherin and epithelial tight junction protein Cldn2. The Snail family of transcription factors has been implicated in cellular acquisition of invasive and migratory properties following repression of the adhesion protein E-cadherin (30–34). In addition, studies have shown the presence of high levels of Snail/Slug protein in invasive mouse and human carcinoma cell lines and tumors in which E-cadherin expression has been lost (35–37). In human breast cancer, Slug was elevated with increasing tumor grade and prognostic indices (33, 38–41). Consistent with these reports, our results in SCID mice provide evidence that that double-transfected cells were highly invasive and metastatic, which could be attributed to inversely expressed snail and E-cad genes. High expression of CXCL12, CXCR4, MMP-2, and MMP-12 in the cyclin D1 cell clone may be responsible for its invasive phenotype (42, 43), although mechanistic studies are required to confirm these findings. Fernandes et al. (42) have shown that MMP-2 expression was regulated by CXCL12, which has also been implicated in cancer cell invasion. The CXCL12/CXCR4 axis has also been implicated in mediating angiogenesis (44) and cell survival (45). Recently, Schmid et al. (46) have shown CXCL12 and CXCR4 expression in human ductal carcinoma in situ and atypical ductal hyperplasia as well as elevated levels in concurrent invasive disease. Our data indicate that introduction of ectopic c-Myc in cyclin D1–transfected cells down-regulates the CXCL12/CXCR4 pathway but up-regulates the Snail2/E-cadherin axis for invasion.

Collectively, the novelty and significance of these results are that overexpression of cyclin D1 was able to transformation of murine mammary epithelial cells from a benign to a malignant status. Additional overexpression of c-myc in cyclin D1–transformed cells makes the tumor more aggressive, as previously hypothesized (11, 24, 47). Cyclin D1 expression alone and cyclin D1/c-myc dual expression may use different molecular pathways to elicit invasion ability.


   Acknowledgments
 
Grant support: NIH grant R01 CA100864 (J.D. Liao) and Susan G. Komen Breast Cancer Foundation grant BCTR02-1648 (J.D. Liao).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Y. Wang and A. Thakur contributed equally to this work.

Current address for Y. Wang: Department of Physiology, Key Lab for Neurodegenerative Disease of Ministry of Education, Capital Medical University, 10 Xitoutiao, Xou Anmen, Fengtai District, Beijing 100054, China.

Received 10/30/06. Revised 2/ 7/07. Accepted 2/14/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gillett C, Fantl V, Smith R, et al. Amplification and overexpression of cyclin D1 in breast cancer detected by immunohistochemical staining. Cancer Res 1994;54:1812–7.[Abstract/Free Full Text]
  2. Fantl V, Stamp G, Andrews A, Rosewell I, Dickson C. Mice lacking cyclin D1 are small and show defects in eye and mammary gland development. Genes Dev 1995;9:2364–72.[Abstract/Free Full Text]
  3. Sicinski P, Donaher JL, Parker SB, et al. Cyclin D1 provides a link between development and oncogenesis in the retina and breast. Cell 1995;82:621–30.[CrossRef][Medline]
  4. Yu Q, Geng Y, Sicinski P. Specific protection against breast cancers by cyclin D1 ablation. Nature 2001;411:1017–21.[CrossRef][Medline]
  5. Wang TC, Cardiff RD, Zukerberg L, Lees E, Arnold A, Schmidt EV. Mammary hyperplasia and carcinoma in MMTV-cyclin D1 transgenic mice. Nature 1994;369:669–71.[CrossRef][Medline]
  6. Geng Y, Whoriskey W, Park MY, et al. Rescue of cyclin D1 deficiency by knockin cyclin E. Cell 1999;97:767–77.[CrossRef][Medline]
  7. Asano K, Sakamoto H, Sasaki H, et al. Tumorigenicity and gene amplification potentials of cyclin D1-overexpressing NIH3T3 cells. Biochem Biophys Res Commun 1995;217:1169–76.[CrossRef][Medline]
  8. Jiang W, Kahn SM, Zhou P, et al. Overexpression of cyclin D1 in rat fibroblasts causes abnormalities in growth control, cell cycle progression and gene expression. Oncogene 1993;8:3447–57.[Medline]
  9. Quelle DE, Ashmun RA, Shurtleff SA, et al. Overexpression of mouse D-type cyclins accelerates G1 phase in rodent fibroblasts. Genes Dev 1993;7:1559–71.[Abstract/Free Full Text]
  10. Han EK, Ng SC, Arber N, Begemann M, Weinstein IB. Roles of cyclin D1 and related genes in growth inhibition, senescence and apoptosis. Apoptosis 1999;4:213–9.[CrossRef][Medline]
  11. Liao DJ, Dickson RB. c-Myc in breast cancer. Endocr Relat Cancer 2000;7:143–64.[Abstract]
  12. Stewart TA, Pattengale PK, Leder P. Spontaneous mammary adenocarcinomas in transgenic mice that carry and express MTV/myc fusion genes. Cell 1984;38:627–37.[CrossRef][Medline]
  13. Jansen-Durr P, Meichle A, Steiner P, et al. Differential modulation of cyclin gene expression by MYC. Proc Natl Acad Sci U S A 1993;90:3685–9.[Abstract/Free Full Text]
  14. Marhin WW, Hei YJ, Chen S, et al. Loss of Rb and Myc activation co-operate to suppress cyclin D1 and contribute to transformation. Oncogene 1996;12:43–52.[Medline]
  15. Philipp A, Schneider A, Vasrik I, et al. Repression of cyclin D1: a novel function of MYC. Mol Cell Biol 1994;14:4032–43.[Abstract/Free Full Text]
  16. Roussel MF, Theodoras AM, Pagano M, Sherr CJ. Rescue of defective mitogenic signaling by D-type cyclins. Proc Natl Acad Sci U S A 1995;92:6837–41.[Abstract/Free Full Text]
  17. Solomon DL, Philipp A, Land H, Eilers M. Expression of cyclin D1 mRNA is not upregulated by Myc in rat fibroblasts. Oncogene 1995;11:1893–7.[Medline]
  18. Pusch O, Soucek T, Wawra E, Hengstschlager-Ottnad E, Bernaschek G, Hengstschlager M. Specific transformation abolishes cyclin D1 fluctuation throughout the cell cycle. FEBS Lett 1996;385:143–8.[CrossRef][Medline]
  19. Qi Y, Tu Y, Yang D, et al. Cyclin a but not cyclin D1 is essential for c-myc-modulated cell-cycle progression. J Cell Physiol 2007;210:63–71.[CrossRef][Medline]
  20. Bouchard C, Dittrich O, Kiermaier A, et al. Regulation of cyclin D2 gene expression by the Myc/Max/Mad network: Myc-dependent TRRAP recruitment and histone acetylation at the cyclin D2 promoter. Genes Dev 2001;15:2042–7.[Abstract/Free Full Text]
  21. Bouchard C, Marquardt J, Bras A, Medema RH, Eilers M. Myc-induced proliferation and transformation require Akt-mediated phosphorylation of FoxO proteins. EMBO J 2004;23:2830–40.[CrossRef][Medline]
  22. Coller HA, Grandori C, Tamayo P, et al. Expression analysis with oligonucleotide microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling, and adhesion. Proc Natl Acad Sci U S A 2000;97:3260–5.[Abstract/Free Full Text]
  23. Dey A, She H, Kim L, et al. Colony-stimulating factor-1 receptor utilizes multiple signaling pathways to induce cyclin D2 expression. Mol Biol Cell 2000;11:3835–48.[Abstract/Free Full Text]
  24. Liao DJ, Natarajan G, Deming SL, et al. Cell cycle basis for the onset and progression of c-Myc-induced, TGFalpha-enhanced mouse mammary gland carcinogenesis. Oncogene 2000;19:1307–17.[CrossRef][Medline]
  25. Thakur A, Xu H, Wang Y, Bollig A, Biliran H, Liao JD. The role of X-linked genes in breast cancer. Breast Cancer Res Treat 2005;93:135–43.[CrossRef][Medline]
  26. Resnitzky D, Gossen M, Bujard H, Reed SI. Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system. Mol Cell Biol 1994;14:1669–79.[Abstract/Free Full Text]
  27. Pagano M, Theodoras AM, Tam SW, Draetta GF. Cyclin D1-mediated inhibition of repair and replicative DNA synthesis in human fibroblasts. Genes Dev 1994;8:1627–39.[Abstract/Free Full Text]
  28. Han EK, Begemann M, Sgambato A, et al. Increased expression of cyclin D1 in a murine mammary epithelial cell line induces p27kip1, inhibits growth, and enhances apoptosis. Cell Growth Differ 1996;7:699–710.[Abstract]
  29. Niu MY, Menard M, Reed JC, Krajewski S, Pratt MA. Ectopic expression of cyclin D1 amplifies a retinoic acid-induced mitochondrial death pathway in breast cancer cells. Oncogene 2001;20:3506–18.[CrossRef][Medline]
  30. Cheng CW, Wu PE, Yu JC, et al. Mechanisms of inactivation of E-cadherin in breast carcinoma: modification of the two-hit hypothesis of tumor suppressor gene. Oncogene 2001;20:3814–23.[CrossRef][Medline]
  31. Elloul S, Elstrand MB, Nesland JM, et al. Snail, Slug, and Smad-interacting protein 1 as novel parameters of disease aggressiveness in metastatic ovarian and breast carcinoma. Cancer 2005;103:1631–43.[CrossRef][Medline]
  32. Yokoyama K, Kamata N, Hayashi E, et al. Reverse correlation of E-cadherin and snail expression in oral squamous cell carcinoma cells in vitro. Oral Oncol 2001;37:65–71.[CrossRef][Medline]
  33. Martin TA, Goyal A, Watkins G, Jiang WG. Expression of the transcription factors snail, slug, and twist and their clinical significance in human breast cancer. Ann Surg Oncol 2005;12:488–96.[Abstract/Free Full Text]
  34. Fearon ER. Connecting estrogen receptor function, transcriptional repression, and E-cadherin expression in breast cancer. Cancer Cell 2003;3:307–10.[CrossRef][Medline]
  35. Batlle E, Sancho E, Franci C, et al. The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol 2000;2:84–9.[CrossRef][Medline]
  36. Cano A, Perez-Moreno MA, Rodrigo I, et al. The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol 2000;2:76–83.[CrossRef][Medline]
  37. Poser I, Dominguez D, de Herreros AG, Varnai A, Buettner R, Bosserhoff AK. Loss of E-cadherin expression in melanoma cells involves up-regulation of the transcriptional repressor Snail. J Biol Chem 2001;276:24661–6.[Abstract/Free Full Text]
  38. Blanco MJ, Moreno-Bueno G, Sarrio D, et al. Correlation of Snail expression with histological grade and lymph node status in breast carcinomas. Oncogene 2002;21:3241–6.[CrossRef][Medline]
  39. Come C, Magnino F, Bibeau F, et al. Snail and slug play distinct roles during breast carcinoma progression. Clin Cancer Res 2006;12:5395–402.[Abstract/Free Full Text]
  40. Sarrio D, Perez-Mies B, Hardisson D, et al. Cytoplasmic localization of p120ctn and E-cadherin loss characterize lobular breast carcinoma from preinvasive to metastatic lesions. Oncogene 2004;23:3272–83.[CrossRef][Medline]
  41. Moody SE, Perez D, Pan TC, et al. The transcriptional repressor Snail promotes mammary tumor recurrence. Cancer Cell 2005;8:197–209.[CrossRef][Medline]
  42. Fernandis AZ, Prasad A, Band H, Klosel R, Ganju RK. Regulation of CXCR4-mediated chemotaxis and chemoinvasion of breast cancer cells. Oncogene 2004;23:157–67.[CrossRef][Medline]
  43. Luker KE, Luker GD. Functions of CXCL12 and CXCR4 in breast cancer. Cancer Lett 2006;238:30–41. Epub 2005 July 25.[CrossRef][Medline]
  44. Wang J, Wang J, Sun Y, et al. Diverse signaling pathways through the SDF-1/CXCR4 chemokine axis in prostate cancer cell lines leads to altered patterns of cytokine secretion and angiogenesis. Cell Signal 2005;17:1578–92.[CrossRef][Medline]
  45. Marchesi F, Monti P, Leone BE, et al. Increased survival, proliferation, and migration in metastatic human pancreatic tumor cells expressing functional CXCR4. Cancer Res 2004;64:8420–7.[Abstract/Free Full Text]
  46. Schmid BC, Rudas M, Rezniczek GA, Leodolter S, Zeillinger R. CXCR4 is expressed in ductal carcinoma in situ of the breast and in atypical ductal hyperplasia. Breast Cancer Res Treat 2004;84:247–50.[CrossRef][Medline]
  47. Liao DJ, Dickson RB. Cell death in MMTV-c- myc transgenic mouse mammary tumors may not be typical apoptosis. Lab Invest 2003;83:1437–49.[CrossRef][Medline]




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