Skip to main content
  • AACR Publications
    • Blood Cancer Discovery
    • Cancer Discovery
    • Cancer Epidemiology, Biomarkers & Prevention
    • Cancer Immunology Research
    • Cancer Prevention Research
    • Cancer Research
    • Clinical Cancer Research
    • Molecular Cancer Research
    • Molecular Cancer Therapeutics

AACR logo

  • Register
  • Log in
  • My Cart
Advertisement

Main menu

  • Home
  • About
    • The Journal
    • AACR Journals
    • Subscriptions
    • Permissions and Reprints
  • Articles
    • OnlineFirst
    • Current Issue
    • Past Issues
    • Rapid Impact Archive
    • Meeting Abstracts
    • Collections
      • COVID-19 & Cancer Resource Center
      • Metabolism Collection
      • Editors' Picks
      • "Best of" Collection
  • For Authors
    • Information for Authors
    • Author Services
    • Best of: Author Profiles
    • Spotlight on Genomic Analysis of Rare and Understudied Cancers
    • Submit
  • Alerts
    • Table of Contents
    • Editors' Picks
    • OnlineFirst
    • Citation
    • Author/Keyword
    • RSS Feeds
    • My Alert Summary & Preferences
  • News
    • Cancer Discovery News
  • COVID-19
  • Webinars
  • Search More

    Advanced Search

  • AACR Publications
    • Blood Cancer Discovery
    • Cancer Discovery
    • Cancer Epidemiology, Biomarkers & Prevention
    • Cancer Immunology Research
    • Cancer Prevention Research
    • Cancer Research
    • Clinical Cancer Research
    • Molecular Cancer Research
    • Molecular Cancer Therapeutics

User menu

  • Register
  • Log in
  • My Cart

Search

  • Advanced search
Molecular Cancer Research
Molecular Cancer Research
  • Home
  • About
    • The Journal
    • AACR Journals
    • Subscriptions
    • Permissions and Reprints
  • Articles
    • OnlineFirst
    • Current Issue
    • Past Issues
    • Rapid Impact Archive
    • Meeting Abstracts
    • Collections
      • COVID-19 & Cancer Resource Center
      • Metabolism Collection
      • Editors' Picks
      • "Best of" Collection
  • For Authors
    • Information for Authors
    • Author Services
    • Best of: Author Profiles
    • Spotlight on Genomic Analysis of Rare and Understudied Cancers
    • Submit
  • Alerts
    • Table of Contents
    • Editors' Picks
    • OnlineFirst
    • Citation
    • Author/Keyword
    • RSS Feeds
    • My Alert Summary & Preferences
  • News
    • Cancer Discovery News
  • COVID-19
  • Webinars
  • Search More

    Advanced Search

Cancer Genes and Genomics

Regulation of Polyamine Analogue Cytotoxicity by c-Jun in Human MDA-MB-435 Cancer Cells1 1 NIH grants P50CA88843 (N. E. D.) and CA51085 (R. A. C.), Breast Cancer Research Foundation grant (N. E. D.), and DOD grant DAMD 17-03-1-0376 (Y. H.).

Yi Huang, Judith C. Keen, Erin Hager, Renee Smith, Amy Hacker, Benjamin Frydman, Aldonia L. Valasinas, Venodhar K. Reddy, Laurence J. Marton, Robert A. Casero Jr. and Nancy E. Davidson
Yi Huang
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Judith C. Keen
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Erin Hager
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Renee Smith
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Amy Hacker
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Benjamin Frydman
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Aldonia L. Valasinas
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Venodhar K. Reddy
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Laurence J. Marton
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Robert A. Casero Jr.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Nancy E. Davidson
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI:  Published February 2004
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Abstract

Several polyamine analogues have efficacy against a variety of epithelial tumor models including breast cancer. Recently, a novel class of polyamine analogues designated as oligoamines has been developed. Here, we demonstrate that several representative oligoamine compounds inhibit in vitro growth of human breast cancer MDA-MB-435 cells. The activator protein-1 (AP-1) transcriptional factor family members, c-Jun and c-Fos, are up-regulated by oligoamines in MDA-MB-435 cells, suggesting a possible AP-1-dependent induction of apoptosis. However, the use of a novel c-Jun NH2-terminal kinase (JNK) inhibitor, SP600125, suggests that inhibition of c-Jun activity sensitized tumor cells to oligoamine-induced cell death. To directly test this hypothesis, cells were stably transfected with the dominant-negative mutant c-Jun (TAM67), which lacks the NH2-terminal transactivation domain. Cells overexpressing TAM67 exhibit normal growth kinetics but demonstrate a significantly increased sensitivity to oligoamine cytotoxicity and attenuated colony formation after oligoamine treatment. Furthermore, oligoamine treatment leads to more profound caspase-3 activation and poly(ADP-ribose) polymerase cleavage in TAM67 transfectants, suggesting that c-Jun acts as an antiapoptosis factor in MDA-MB-435 cells in response to oligoamine treatment. These findings indicate that oligoamine-inducible AP-1 plays a prosurvival role in oligoamine-treated MDA-MB-435 cells and that JNK/AP-1 might be a potential target for enhancing the therapeutic efficacy of polyamine analogues in human breast cancer.

Introduction

The polyamines (putrescine, spermidine, and spermine) are naturally occurring polycationic alkylamines that are required for cell growth. Because of the critical role of polyamines in the regulation of cell growth, the polyamine metabolic pathway is an attractive target for antineoplastic strategies (1–3). The primary regulatory enzymes of polyamine biosynthesis include ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase, spermidine synthase, and spermine synthase. The enzymes spermidine/spermine N1-acetyltransferase (SSAT) and spermine oxidase also play a significant rate-limiting role in polyamine catabolism (4–6). The best characterized polyamine biosynthetic pathway inhibitor is α-difluoromethylornithine, an irreversible inhibitor of ODC, which inhibits tumor cell growth in preclinical models, has been tested in phase I/II clinical trials, and is currently being examined as a chemopreventive agent (7–9).

Based on the feedback mechanisms of natural polyamines, whereby they regulate their own synthesis, another useful strategy to develop polyamine metabolism inhibitors is the use of polyamine analogues that can mimic some of the regulatory roles of natural polyamines but are unable to replace the actual function of natural polyamines for cell growth (10–12). Several polyamine analogues have shown antineoplastic activity in a variety of tumor types. For example, N1,N11-diethylnorspermine inhibits polyamine biosynthesis, decreases polyamine transport, and greatly increases SSAT activity in several tumor models (13, 14). Recently, a series of novel oligoamine analogues have been synthesized (15). Some of these new analogues have shown significant inhibitory activity against in vitro and in vivo tumor cell growth (16, 17).

In our previous studies, several early polyamine analogues were reported to induce programmed cell death in human breast cancer cells, but the cell death mechanisms have been uncertain (18). For example, N1,N11-diethylnorspermine-related superinduction of SSAT with subsequent depletion of natural polyamine pools was noted in specific human breast cancer cell lines (19), a finding that has been confirmed by conditional overexpression of SSAT in breast cancer MCF-7 cells (20). Although superinduction of SSAT seems to be directly associated with growth inhibition by some polyamine analogues, other polyamine analogues do not highly induce SSAT but can still inhibit tumor cell growth (21, 22). These results suggest that polyamine analogue-induced cell death occurs through multiple agent-specific and cell type-specific mechanisms. Several important growth-associated or cell cycle-associated genes or pathways have been reported to be affected by specific polyamine analogues (17, 23, 24). However, little is known thus far about the exact mechanisms by which other polyamine analogues inhibit growth and induce apoptosis in tumor cells.

An important mediator that is widely involved in regulating cellular proliferation, differentiation, and apoptosis is the activator protein-1 (AP-1) transcription factor family (25). AP-1 proteins are homodimers or heterodimers composed of leucine zipper proteins that belong to the Jun (c-Jun, JunB, and JunD), Fos (c-Fos, FosB, Fra-1, and Fra-2), Jun dimerization partners (JDP1 and JDP2), and activating transcription factors (ATF2, LRF1/ATF3, and B-ATF) subgroups (26, 27). The major upstream regulator of AP-1 is the c-Jun NH2-terminal kinase (JNK), one subgroup of the mitogen-activated protein kinase (MAPK) family. After exposure of cells to several cytokines or extracellular stresses, JNK is activated and subsequently phosphorylates its major target, c-Jun, and related molecules. This in turn leads to the enhanced transcriptional regulation of c-Jun and its dimerization partners with subsequent effects on the promoter regions of downstream target genes (27).

In our previous work, we noted that a new oligoamine analogue, SL-11144, inhibited growth and induced apoptosis in several human breast cancer cell lines (17). In some cases, this was associated with induction of the Jun protein. In this study, we have examined the mechanisms of cytotoxicity of several oligoamines using the human MDA-MB-435 cancer cell line as a model system. Our data demonstrate that these oligoamines significantly inhibit cell growth and activate AP-1 members, c-Jun and c-Fos. By using a selective JNK inhibitor and the stable transfection of dominant-negative mutant c-Jun (TAM67), we provide evidence that c-Jun plays a protective role in oligoamine-induced cell death in MDA-MB-435 cells.

Results

Growth Inhibition by Oligoamines

The MDA-MB-435 cells were chosen as a model system as they exhibit highly metastatic and aggressive behavior in vivo compared with other human breast cancer cell lines (28–30). The in vitro sensitivities of MDA-MB-435 cells to SL-11144, SL-11159, and SL-11172 (Fig. 1 ) were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) growth inhibition assay. Tumor cells were treated with increasing concentrations of oligoamines for 24 and 48 h, respectively. All three agents showed significant growth inhibitory effects against MDA-MB-435 cells in a time- and dose-dependent manner (Fig. 2 ).

FIGURE 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 1.

Struct ures of natural polyamines and oligoamines SL11144, SL-11159, and SL-11172.

FIGURE 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 2.

Oligoamines inhibit growth of human breast cancer MDA-MB-435 cells in a time- and dose-dependent manner. MDA-MB-435 cells were treated with increasing concentrations of SL-11144, SL-11159, or SL-11172 for 24 or 48 h. MTT assays were performed as described in “Materials and Methods.” Points, means of three independent experiments performed in quadruplicate; bars, SD.

Oligoamines Up-Regulate c-Jun and c-Fos

AP-1 is an important signaling complex in the regulation of cellular proliferation, differentiation, apoptosis, and metastasis (31). Therefore, we first examined the impact of oligoamines on the expression of AP-1 family members, c-Jun and c-Fos, in MDA-MB-435 cells. All three oligoamines significantly induced the phosphorylation of c-Jun and enhanced the protein level of both c-Jun and c-Fos in a dose-dependent manner within 24 h of treatment (Fig. 3 ). The phosphorylation of c-Jun can occur within 6 h of oligoamine exposure (data not shown). These results suggested that up-regulation of the AP-1 family might play an active role in the mediation of oligoamine-induced growth inhibition in MDA-MB-435 cells.

FIGURE 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 3.

Effects of oligoamines on c-Jun and c-Fos. MDA-MB-435 cells were treated with 1, 5, and 10 μm of SL-11144, SL-11159, or SL-11172 for 24 h. Equal amounts of protein (50 μg/lane) were fractionated on 12% SDS-PAGE gels and transferred to PVDF membranes followed by immunoblotting with anti-c-Jun and anti-c-Fos polyclonal antibodies and analysis as described in “Materials and Methods.” Actin protein was blotted as a control. Each experiment was repeated twice with similar results.

SP600125 Blocks c-Jun Activation and Promotes the Cytotoxicity of Oligoamine

SP600125 is a novel selective JNK1, JNK2, and JNK3 inhibitor that can inhibit the phosphorylation of c-Jun (32, 33). To examine whether the inhibition of c-Jun phosphorylation could affect oligoamine-induced cell death, MDA-MB-435 cells were treated with SP600125 or oligoamines alone or simultaneously. By Western blot, we determined that cotreatment with oligoamines and SP600125 significantly blocked the phosphorylation and decreased the expression of c-Jun compared with that seen with oligoamine treatment alone (Fig. 4A ). MTT analysis of the effects of the c-Jun phosphorylation inhibitor on oligoamine-induced cell growth inhibition indicated that simultaneous treatment with SP600125 and oligoamine resulted in increased cytotoxicity compared with that seen with either agent alone (Fig. 4B). It is important to note that SP600125 treatment does not affect accumulation of SL-11144 in MDA-MB-435 cells (data not shown).

FIGURE 4.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 4.

SP600125 blocks c-Jun activation and promotes the cytotoxicity of oligoamine. A. MDA-MB-435 cells were treated with 10 μm SL-11144, SL-11159, or SL-11172 with or without the cotreatment with 10 μm SP600125 for 24 h. Equal amounts of cellular protein (50 μg/lane) were fractionated on 12% SDS-PAGE gels and transferred to PVDF membranes followed by immunoblotting with anti-c-Jun polyclonal antibodies and analysis as described in “Materials and Methods.” Actin protein was blotted as a control. Each experiment was repeated twice with similar results. B. MDA-MB-435 cells were treated with 10 μm SL-11144, SL-11159, or SL-11172 with or without the cotreatment with 10 μm SP600125 for 24 h. MTT assays were performed as described in “Materials and Methods.” Columns, means of three independent experiments performed in quadruplicate; bars, SD. **, P < 0.01; ***, P < 0.001, statistically significant differences using Student's t test.

Overexpression of TAM67 Sensitizes Tumor Cells to Oligoamine Cytotoxicity

The finding that SP600125 blocks JNK/c-Jun pathway activation and increases the cytotoxicity of oligoamines suggests that the transcription factor c-Jun may exert a protective role in oligoamine-induced cell death. However, further evidence is needed to support this hypothesis because JNK activation may concurrently regulate a variety of other downstream genes or pathways with diverse functions (27). To further dissect the effect of c-Jun activity on oligoamine-induced cell death, a vector expressing the c-Jun dominant-negative mutant, TAM67, was constructed and stably transfected into MDA-MB-435 cells. Wild-type c-Jun can be phosphorylated by JNK at two serine residues (Ser63 and Ser73) proximal to the transactivation domain, which is required for the efficient transactivation of c-Jun (34, 35). TAM67 is a mutant form of c-Jun in which the transactivation domain (amino acids 3–122) has been deleted, leaving the COOH-terminal DNA binding and dimerization domain intact. Such a mutant c-Jun protein is unable to activate its target genes but still possesses the ability to bind to AP-1 site in promoter regions of target genes and competitively quench the transactivation activity of endogenous wild-type Jun and its dimerization partners (36, 37). Three single clones (TAM67-12, TAM67-19, and TAM67-21) that highly express the ~29-kDa dominant-negative mutant c-Jun protein were identified (Fig. 5A ), and these three clones were used for a series of functional experiments. We first examined the effect of TAM67 on the general growth rate of MDA-MB-435 transfectants. Cell growth over 96 h was similar for the parental wild-type, vector, and TAM67 mutant cell lines (Fig. 5B), indicating that the stable expression of either empty vector or TAM67 does not adversely affect the growth of MDA-MB-435 cells.

FIGURE 5.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 5.

Characterization of TAM67 transfectants. A. MDA-MB-435 cells were stably transfected with pcDNA3.1-TAM67 as described in “Materials and Methods.” Protein isolated from parental cells, empty vector transfectants, and TAM67 transfected single colonies were subjected to immunoblotting with an antibody to the COOH terminus of c-Jun to detect TAM67 at 29 kDa. Three geneticin-resistant clones (TAM67-12, TAM67-19, and TAM67-21) were shown to express high level of TAM67. Endogenous Jun proteins appeared at the upper region of the gel around 39 kDa. B. Proliferation rates of parental cells, empty vector transfectants, and three independent TAM67 stable transfectants were measured as described in “Materials and Methods.” Points, averages of three independent experiments performed in triplicate; bars, SD.

TAM67 transfected clones were then compared to determine whether the expression of the dominant-negative mutant c-Jun altered the response of tumor cells to oligoamines. The vector control clone and the three independent dominant-negative mutant c-Jun transfectants were treated with increasing concentrations of SL-11144, SL-11159, or SL-11172 (1–20 μm) for 24 h and analyzed by MTT. When compared with the vector control, all three TAM67 clones were significantly more sensitive to treatment with SL-11144 and SL-11159 at all doses studied (P < 0.001). The TAM67 transfectants were also more sensitive to lower concentrations of SL-11172 treatment (1–2.5 μm, P < 0.001; Fig. 6 ). However, at higher concentrations (5.0 μm or higher) of SL-11172 treatment, there was no obvious difference in cellular sensitivity to SL1-11172 between controls and transfectants.

FIGURE 6.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 6.

Overexpression of TAM67 sensitizes tumor cells to oligoamine cytotoxicity. Cells transfected with empty pcDNA3.1 vectors (Vector) and TAM67 cDNA transfected clones (TAM67-12, TAM67-19, and TAM67-21) were treated with 1, 2.5, 5, or 10 μm of SL-11144, SL-11159, and SL-11172 for 24 h. MTT assays were performed as described in “Materials and Methods.” Points, means of three independent experiments performed in quadruplicate; bars, SD. P < 0.001, statistically significant differences using Student's t test between transfected cells and parental or vector transfected cells treated by SL-11144 (2.5–20 μm), SL-11159 (2.5–20 μm), and SL11172 (1.0 and 2.5 μm).

To confirm the increased sensitivity of TAM67 transfectants to oligoamines, vector and TAM67 transfected cells were subjected to colony formation analysis. Cells were treated with 0.1 μm of each oligoamine for 12 h. The colony-forming efficiency of all three TAM67 transfected cell lines was dramatically diminished after treatment with all three oligoamines compared with the vector transfectant (Fig. 7 ). These results suggest that specific inhibition of transactivation of endogenous wild-type c-Jun significantly increases the sensitivity of MDA-MB-435 cells to oligoamine cytotoxicity.

FIGURE 7.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 7.

Effect of TAM67 transfection on colony formation. Vector and TAM67 transfected MDA-MB-435 cells were treated with 0.1 μm oligoamines (SL-11144, SL-11159, or SL-11172) for 12 h. Cells were washed, medium was replaced with drug-free medium, and cells were incubated for 8 days. Positive colonies were counted as described in “Materials and Methods.” Results are average percentages of relative colony formation efficiency (numbers of colonies in treated groups/numbers of colonies in untreated groups). Columns, means of three independent experiments plated in triplicate; bars, SD.

TAM67 Overexpression Enhances Oligoamine-Induced Apoptosis

In our recent studies, oligoamines have been demonstrated to activate apoptosis-related pathways and induce apoptotic cell death in human breast cancer cells (17). To investigate if overexpression of dominant-negative mutant c-Jun could affect apoptotic response to oligoamines in MDA-MB-435 cells, expression of two key apoptosis effectors, caspase-3 and poly(ADP-ribose) polymerase (PARP), was examined by Western blotting. The vector transfectant and a representative dominant-negative mutant c-Jun transfectant (TAM67-21) were treated with SL-11144 for 24 h. SL-11144 treatment led to a greater decrease in the caspase-3 level and more significant cleavage of the PARP protein in TAM67-21 cells compared with the vector control clone (Fig. 8 ). These results indicate that inhibition of c-Jun activation may enhance apoptotic cell death induced by oligoamines in MDA-MB-435 cells.

FIGURE 8.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 8.

Effects of TAM67 on oligoamine-induced apoptosis. Parental, vector, and TAM67 transfected MDA-MB-435 cells were treated with 10 μm SL-11144 for 24 h. Equal amounts of protein (50 μg/lane) were fractionated on 12% SDS-PAGE gels and transferred to PVDF membranes followed by immunoblotting with anti-caspase-3 and anti-PARP monoclonal or polyclonal antibodies and analysis as described in “Materials and Methods.” Actin protein was blotted as a control. Each experiment was repeated twice with similar results.

Effects of Oligoamine on Intracellular Polyamines Metabolism in TAM67 and Vector Control Transfectants

To address whether the overexpression of TAM67 alters the effects of oligoamines on the polyamine metabolic pathway, we evaluated the intracellular polyamine pools and the activities of ODC, a key polyamine biosynthetic enzyme, and SSAT, a key polyamine catabolic enzyme, in empty vector and TAM67 transfected cells. The TAM67-21 transfectant was again selected as representative for these studies. Treatment of both vector controls and TAM67-21 transfectants with 10 μm SL-11144 for 12 h was associated with a similar decrease in natural polyamine levels, increased SSAT activity, and diminished ODC activity in both cell lines (Table 1).

View this table:
  • View inline
  • View popup
Table 1.

Effects of Oligoamine on Polyamine Pools, SSAT, and ODC Activities in Empty Vector and TAM67 Transfected Cells

Discussion

The antiproliferative mechanisms of various polyamine analogues are being elucidated. Possible mechanisms include the fact that polyamine analogues can bind to nucleic acids, leading to the distortion of nucleic acid structure and impairment of normal function (38). Another potential mechanism is the rapid depletion of the natural polyamine pool associated with the superinduction of SSAT by polyamine analogues in some cell lines (19, 20, 22, 39). The oligoamines have longer chains than natural mammalian cellular polyamine molecules, which allow the analogues to condense and collapse DNA at much lower concentrations (15, 16).

In this work, we demonstrate that the decamines, SL-11144 and SL-11159, and the dodecamine, SL-11172, significantly inhibit the in vitro growth of human breast cancer MDA-MB-435 cells and increase c-Jun and c-Fos expression and c-Jun phosphorylation (Fig. 3). Cotreatment with a JNK selective inhibitor, SP600125, sensitizes tumor cells to the oligoamines. This result implicates a possible protective role of the JNK/Jun pathway in oligoamine-treated MDA-MB-435 cells. AP-1 family members, particularly c-Jun and c-Fos, play critical roles in cellular proliferation, differentiation, transformation, and apoptosis, likely through their effects on several important AP-1 target genes, including cyclin D1, p16, p19Arf, p53, p21cip1/waf1, and Fas L (40–43). Due to the complexity of regulatory signaling impinging on AP-1/c-Jun, the net effect of AP-1 on the balance between cell survival and death varies greatly, depending on cell context, nature of the extracellular stimuli, and the signaling pathways that are simultaneously activated. For example, studies in neuronal cells demonstrated that inhibition of c-Jun activity by dominant-negative mutant forms protects cells from apoptosis induced by withdrawal of nerve growth factor, and expression of a phosphorylation-deficient c-Jun mutant blocked phosphorylation of c-Jun by JNK and inhibited apoptosis (44–48). However, other lines of evidence suggest that JNK/Jun activation may protect other cells from stress-induced death. For example, in vitro studies demonstrated that JNK1 plays a protective role in Fas-induced apoptosis (49), and cells overexpressing mutant c-Jun are more vulnerable to apoptosis triggered by UV irradiation (50). Other in vivo studies suggested that c-Jun-deficient embryos exhibited massive apoptosis in liver cells (51). Taken together, these results suggest that AP-1 may have divergent functions in regulating stress-mediated cell death in different cell contexts.

To study the exact role of c-Jun in oligoamine-induced cell growth inhibition and death, clones of the MDA-MB-435 cell line that stably express a mutant transactivation domain deletion c-Jun (TAM67) were established. All three transfectants displayed normal growth, suggesting that dominant-negative mutant c-Jun protein is well tolerated in untreated cells. However, MTT proliferation assays show that mutant c-Jun transfectants were significantly more sensitive to the oligoamines over a broad range of concentrations. In addition, TAM67 transfectants exhibited dramatically reduced clonogenic efficiency following oligoamine exposure. These results indicate that c-Jun is a stress response protein and cell survival promoter in MDA-MB-435 cells. It is interesting to note that transfectants showed increased susceptibility to the dodecamine analogue, SL-11172, only with low-dose treatment (<5 μm). One possible explanation is that, structurally, SL-11172 has a longer chain than the decamines, SL-11144 and SL-11159, and therefore exhibits stronger DNA binding ability and cellular toxicity. Higher doses of SL-11172 induce more significant expression and phosphorylation of endogenous c-Jun than SL-11144 or SL-11159 (Fig. 3); thus, the level of TAM67 expression may not be sufficient to suppress the protective activities of endogenous c-Jun induced by higher doses of SL-11172. Previous studies in our laboratory demonstrated that oligoamines can induce apoptotic cell death in MDA-MB-435 cells through activation of critical death effectors like caspase-3 and PARP (17). The current study revealed that oligoamine produces a more profound caspase-3 activation and PARP cleavage in TAM67 transfectants than in control cells, suggesting that c-Jun acts as an antiapoptosis factor in MDA-MB-435 cells in response to oligoamine treatment.

Sensitization to oligoamine-induced cell death by TAM67 transfection has also been observed in another human breast cancer cell line, MCF-7. A clone of MCF-7 cells transfected with TAM67 exhibited a similar increase in sensitivity to oligoamines and enhanced p53/p21waf1/cip1 pathway activation under oligoamine treatment (data not shown). In addition, activation of the MAPK pathway by a polyamine analogue was recently demonstrated to play a protective role in MALME-3M melanoma cells (23). All these results suggest that activation of the MAPK pathway and its downstream effector, AP-1, may play an important protective role in certain types of tumor cells in response to specific polyamine analogue-induced cell death.

Our studies also investigated the effects of oligoamine on the polyamine metabolic pathway in vector and TAM67 transfected cells (Table 1). ODC is a key regulatory enzyme in polyamine biosynthesis. The significant decrease of ODC activity in SL-11144-treated MDA-MB-435 cells suggests that ODC might be one of the targets for oligoamines. However, it should be noted that the effects of SL-11144 on ODC activity appear to be indirect. When cellular extracts of MDA-MB-435 cells containing ODC enzyme are exposed to concentrations of SL-11144 up to 1000 μm, no change in ODC activity is observed. This is in contrast to results observed with the specific ODC inhibitor, 2-difluoromethylornithine, which produces nearly complete inhibition of ODC activity in cell extracts at concentrations > 100 μm (data not shown). Likely, the cellular effects of SL-11144 resulting in decreased ODC activity are not a result of direct enzyme inhibition but are a result of regulatory mechanisms including the increased production of ODC antizyme as demonstrated by Mitchell et al. (52).

In conclusion, we present evidence that treatment with a JNK inhibitor or specific blockade of AP-1 by a dominant-negative mutant c-Jun sensitizes MDA-MB-435 cells to cell death induced by three novel polyamine analogues. The results strongly suggest that the AP-1 proteins play a protective role in this cell line in response to stress and cell death signals. Our work provides new insights into the molecular mechanisms of polyamine analogues in cancer cells. It also suggests that AP-1 may be a useful target for improving the therapeutic efficacy of polyamine analogues in human breast cancer.

Materials and Methods

Compounds, Cell Line, and Culture Conditions

The polyamine analogues SL-11144, SL-11159, and SL-11172 (Fig. 1) were synthesized as reported previously (15, 16). A concentrated stock solution (10 mm in double-distilled water) was diluted with the medium to the desired concentrations for specific experiments. The selective JNK1, JNK2, and JNK3 inhibitor SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one) from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA) was prepared as a stock solution of 20 mm in 100% DMSO. The MDA-MB-435 human carcinoma cell line was originally isolated from the pleural effusion of a patient with breast carcinoma (28, 29). It should be noted that one recent report, using microarray analysis, indicated that MDA-MB-435 cells expressed certain markers associated with melanoma (53). However, this report has not been confirmed and is not consistent with the clinical course of the patient from whom the cells were derived. Cells were maintained in improved MEM supplemented with 5% fetal bovine serum, 2 mm glutamine, and 100 units/ml penicillin/streptomycin and incubated at 37°C in a 5% CO2 atmosphere.

Construction of Expression Plasmid and Stable Transfection of TAM67

The cDNA of mutant c-Jun (TAM67) that lacks amino acids 3–122 of the transactivation domain was kindly provided by Dr. Steve N. Georas (Johns Hopkins) and subcloned into the pcDNA3.1 mammalian expression vector (Invitrogen, Carlsbad, CA). Transfections were performed by LipofectAMINE Plus reagent (Invitrogen) as recommended by the manufacturer. Stable transfectants were selected by incubating the cells in the medium containing 500 μg/ml geneticin (G418). Cells from individual colonies were examined for TAM67 expression by Western blot analysis with an antibody that recognizes the COOH terminus of c-Jun (sc-44; Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Growth Inhibition and Colony Formation Assays

Growth inhibition was assessed by MTT assays as described previously (54). Briefly, 2000–5000 cells were plated in 96-well dishes and treated with the various concentrations of drug regimes for the indicated times. All of the experiments were plated in quadruplicate and were carried out at least thrice. The results of assays were presented as means ± SD. Colony formation assay was performed as published previously (55). Colonies that contained >50 cells were scored. Relative clonogenic efficiency was assessed as numbers of colonies in treated group/numbers of colonies in control group. All experiments were plated in triplicate and were carried out at least thrice. The results were expressed as means ± SD.

Western Blotting

Cells were treated with indicated drug concentrations and times, harvested by trypsinization, and washed with PBS. Cellular protein was isolated using a protein extraction buffer containing 150 mm NaCl, 10 mm Tris (pH 7.2), 5 mm EDTA, 0.1% Triton X-100, 5% glycerol, and 2% SDS. Protein concentrations were determined using Micro Protein Assay Kit (Pierce Chemical Co., Rockford, IL). Equal amounts of protein (50 μg/lane) were fractionated on 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with primary antibodies against c-Jun, c-Fos, caspase-3 (1:2000; Santa Cruz Biotechnology), and PARP (1:2000; Calbiochem, San Diego, CA). After washing with PBS, the membranes were incubated with peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibody (1:3000; DAKO Corp., Carpinteria, CA) followed by enhanced chemiluminescent staining using the enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ). Actin was used to normalize for protein loading. All the experiments were performed at least twice with similar results.

Analysis of Intracellular Polyamine Pools, SSAT Activity, and ODC Activity

The intracellular polyamine content of treated and untreated cells was determined by precolumn dansylation and reversed phase high-performance liquid chromatography (56). SSAT and ODC activities were measured using cellular extracts as described previously (57, 58). Protein concentrations were determined according to the method of Bradford (59).

Acknowledgments

We thank Dr. Steve N. Georas (Johns Hopkins) for the cDNA of mutant c-Jun (TAM67).

Footnotes

  • ↵1 NIH grants P50CA88843 (N. E. D.) and CA51085 (R. A. C.), Breast Cancer Research Foundation grant (N. E. D.), and DOD grant DAMD 17-03-1-0376 (Y. H.).

    • Accepted December 23, 2003.
    • Received October 21, 2003.
    • Revision received December 15, 2003.
  • American Association for Cancer Research

References

  1. ↵
    Pegg AE. Polyamine metabolism and its importance in neoplastic growth and a target for chemotherapy. Cancer Res 1988; 48: 759–74.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    Porter CW, Herrera-Ornelas L, Pera P, et al. Polyamine biosynthetic activity in normal and neoplastic human colorectal tissues. Cancer 1987;60:1275–81.
    OpenUrlCrossRefPubMed
  3. ↵
    Davidson NE, Mank AR, Prestigiacomo LJ, et al. Growth inhibition of hormone-responsive and -resistant human breast cancer cells in culture by N1,N12-bis(ethyl)spermine. Cancer Res 1993;53:2071–5.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Casero RA Jr, Celano P, Ervin SJ, et al. Isolation and characterization of a cDNA clone that codes for human spermidine/spermine N1-acetyltransferase. J Biol Chem 1991;266:810–4.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Wang Y, Devereux W, Woster PM, et al. Cloning and characterization of the human polyamine oxidase that is Inducible by polyamine analogue exposure. Cancer Res 2001;61:5370–3.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Casero RA Jr, Woster PM. Terminally alkylated polyamine analogues as chemotherapeutic agents. J Med Chem 2001;44:1–26.
    OpenUrlCrossRefPubMed
  7. ↵
    Meyskens FL Jr, Gerner EW. Development of difluoromethylornithine (DFMO) as a chemoprevention agent. Clin Cancer Res 1999;5:945–51.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Gupta S, Ahmad N, Marengo SR, et al. Chemoprevention of prostate carcinogenesis by α-difluoromethylornithine in TRAMP mice. Cancer Res 2000;60:5125–33.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    O'Shaughnessy JA, Demers LM, Jones SE, et al. α-Difluoromethylornithine as treatment for metastatic breast cancer patients. Clin Cancer Res 1999;5:3438–44.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Bergeron RJ, Neims AH, McManis JS, et al. Synthetic polyamine analogues as antineoplastics. J Med Chem 1988;31:1183–90.
    OpenUrlCrossRefPubMed
  11. ↵
    Marton LJ, Pegg AE. Polyamines as targets for therapeutic intervention. Annu Rev Pharmacol Toxicol 1995;35:55–91.
    OpenUrlCrossRefPubMed
  12. ↵
    Casero RA Jr, Pegg AE. Spermidine/spermine N1-acetyltransferase—the turning point in polyamine metabolism. FASEB J 1993;7:653–61.
    OpenUrlAbstract
  13. ↵
    Casero RA Jr, Mank AR, Xiao L, et al. Steady-state messenger RNA and activity correlates with sensitivity to N1,N12-bis(ethyl)spermine in human cell lines representing the major forms of lung cancer. Cancer Res 1992;52:5359–63.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Casero RA Jr, Mank AR, Saab NH, et al. Growth and biochemical effects of unsymmetrically substituted polyamine analogues in human lung tumor cells. Cancer Chemother Pharmacol 1995;36:69–74.
    OpenUrlCrossRefPubMed
  15. ↵
    Bacchi CJ, Weiss LM, Lane S, et al. Novel synthetic polyamines are effective in the treatment of experimental microsporidiosis, an opportunistic AIDS-associated infection. Antimicrob Agents Chemother 2002;46:55–61.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Valasinas A, Reddy VK, Blokhin AV, et al. Long-chain polyamines (oligoamines) exhibit strong cytotoxicities against human prostate cancer cells. Bioorg Med Chem 2003;11:4121–31.
    OpenUrlCrossRefPubMed
  17. ↵
    Huang Y, Hager ER, Phillips DL, et al. A novel polyamine analog inhibits growth and induces apoptosis in human breast cancer cells. Clin Cancer Res 2003;9:2769–77.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    McCloskey DE, Casero RA, Woster PM, et al. Induction of programmed cell death in human breast cancer cells by an unsymmetrically alkylated polyamine analogue. Cancer Res 1995;55:3233–6.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Davidson NE, Hahm HA, McCloskey DE, Woster PM, Casero RA Jr. Clinical aspects of cell death in breast cancer: the polyamine pathway as a new target for treatment. Endocr Relat Cancer 1999;6:69–73.
    OpenUrlAbstract
  20. ↵
    Vujcic S, Halmekyto M, Diegelman P, et al. Effects of conditional overexpression of spermidine/spermine N1-acetyltransferase on polyamine pool dynamics, cell growth, and sensitivity to polyamine analogs. J Biol Chem 2000;275:38319–28.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Webb HK, Wu Z, Sirisoma N, et al. 1-(N-alkylamino)-11-(N-ethylamine)-4,8-diazaundecanes: simple synthetic polyamine analogues that differentially alter tubulin polymerization. J Med Chem 1999;42:1415–21.
    OpenUrlCrossRefPubMed
  22. ↵
    Chen Y, Kramer DL, Diegelman P, et al. Apoptotic signaling in polyamine analogue-treated SK-MEL-28 human melanoma cells. Cancer Res 2001;61:6437–44.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Chen Y, Alm K, Vujcic S, et al. The role of mitogen-activated protein kinase activation in determining cellular outcomes in polyamine analogue-treated human melanoma cells. Cancer Res 2003;63:3619–25.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Kramer DL, Vujcic S, Diegelman P, et al. Polyamine analogue induction of the p53-p21WAF1/CIP1-Rb pathway and G1 arrest in human melanoma cells. Cancer Res 1999;59:1278–86.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Angel P, Karin M. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochem Biophya Acta 1991;1072:129–57.
    OpenUrl
  26. ↵
    Chimenov Y, Kerppola TK. Close encounters of many kinds: Fos-Jun interactions that mediate transcription regulatory specificity. Oncogene 2001;6:533–42.
    OpenUrl
  27. ↵
    Weston CR, Davis RJ. The JNK signal transduction pathway. Curr Opin Genet Dev 2002;12:14–21.
    OpenUrlCrossRefPubMed
  28. ↵
    Zhang RD, Fidler IJ, Price JE. Relative malignant potential of human breast carcinoma cell lines established from pleural effusion and a brain metastasis. Invasion Metastasis 1991;11:204–15.
    OpenUrlPubMed
  29. ↵
    Price JE, Polyzos A, Zhang RD, et al. Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice. Cancer Res 1990;50:717–21.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Glinsky GV, Glinsky VV, Ivanova AB, et al. Apoptosis and metastasis: increased apoptosis resistance of metastatic cancer cells is associated with the profound deficiency of apoptosis execution mechanisms. Cancer Lett 1997;115:185–93.
    OpenUrlCrossRefPubMed
  31. ↵
    Gee JM, Barroso AF, Ellis IO, et al. Biological and clinical associations of c-jun activation in human breast cancer. Int J Cancer 2000;89:177–86.
    OpenUrlCrossRefPubMed
  32. ↵
    Bennett BL, Sasaki DT, Murray BW, et al. SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc Natl Acad Sci 2001;98:13681–6.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Shin M, Yan C, Boyd D. An inhibitor of c-jun aminoterminal kinase (SP600125) represses c-Jun activation, DNA-binding and PMA-inducible 92-kDa type IV collagenase expression. Biochim Biophys Acta 2002;1589:311–6.
    OpenUrlPubMed
  34. ↵
    Pulverer B, Kyriakis J, Avruch J, et al. Phosphorylation of c-jun by MAP kinases. Nature 1991;353:670–4.
    OpenUrlCrossRefPubMed
  35. ↵
    Franklin CC, Sanchez V, Wagner F, et al. Phorbol ester-induced amino terminal phosphorylation of c-Jun but not JunB regulates transcriptional activation. Proc Natl Acad Sci 1992;89:7247–51.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Alani R, Brown P, Binetruy B, et al. The transactivating domain of the c-Jun proto-oncoprotein is required for cotransformation of rat embryo cells. Mol Cell Biol 1991;11:6286–95.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Brown PH, Alani R, Preis LH, et al. Suppression of oncogene-induced transformation by a deletion mutant of c-jun. Oncogene 1993;8:877–86.
    OpenUrlPubMed
  38. ↵
    Feuerstein BG, Williams LD, Basu HS, et al. Implications and concepts of polyamine-nucleic acid interactions. J Cell Biochem 1991;46:37–47.
    OpenUrlCrossRefPubMed
  39. ↵
    McCloskey DE, Yang J, Woster PM, et al. Polyamine analogue induction of programmed cell death in human lung tumor cells. Clin Cancer Res 1996;2:441–6.
    OpenUrlAbstract
  40. ↵
    Shaulian E, Karin M. AP-1 in cell proliferation and survival. Oncogene 2001;20:2390–400.
    OpenUrlCrossRefPubMed
  41. ↵
    Albanese C, Johnson J, Watanabe G, et al. Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions. J Biol Chem 1995;270:23589–97.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    Passegue E, Wagner EF. JunB suppresses cell proliferation by transcriptional activation of p16 (INK4a) expression. EMBO J 2000;19:2969–79.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    Schreiber M, Kolbus A, Piu F, et al. Control of cell cycle progression by c-Jun is p53 dependent. Genes Dev 1999;13:607–19.
    OpenUrlAbstract/FREE Full Text
  44. ↵
    Estus S, Zaks WJ, Freeman RS, et al. Altered gene expression in neurons during programmed cell death: identification of c-jun as necessary for neuronal apoptosis. J Cell Biol 1994;127:1717–27.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    Ham J, Babij C, Whitfield J, et al. A c-Jun dominant negative mutant protects sympathetic neurons against programmed cell death. Neuron 1995;14:927–39.
    OpenUrlCrossRefPubMed
  46. ↵
    Xia Z, Dickens M, Raingeaud J, et al. Opposing Effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 1995;270:1326–31.
    OpenUrlAbstract/FREE Full Text
  47. ↵
    Le-Niculescu H, Bonfoco E, Kasuya Y, et al. Withdrawal of survival factors results in activation of the JNK pathway in neuronal cells leading to Fas ligand induction and cell death. Mol Cell Biol 1999;19:751–63.
    OpenUrlAbstract/FREE Full Text
  48. ↵
    Watson A, Eilers A, Lallemand D, et al. Phosphorylation of c-Jun is necessary for apoptosis induced by survival signal withdrawal in cerebellar granule neurons. J Neurosci 1998;18:751–62.
    OpenUrlAbstract/FREE Full Text
  49. ↵
    Nishina H, Fischer KD, Radvanyi L, et al. Stress-signaling kinase Sek1 protects thymocytes from apoptosis mediated by CD95 and CD3. Nature 1997;385:350–3.
    OpenUrlCrossRefPubMed
  50. ↵
    Wisdom R, Johnson RS, Moore C. c-Jun regulates cell cycle progression and apoptosis by distinct mechanisms. EMBO J 1999;18:188–97.
    OpenUrlAbstract
  51. ↵
    Eferl R, Sibilia M, Hilberg F, et al. Functions of c-Jun in liver and heart development. J Cell Biol 1999;145:1049–61.
    OpenUrlAbstract/FREE Full Text
  52. ↵
    Mitchell JL, Leyser A, Holtorff MS, et al. Antizyme induction by polyamine analogues as a factor of cell growth inhibition. Biochem J 2002;366:663–71.
    OpenUrlCrossRefPubMed
  53. ↵
    Ellison G, Klinowska T, Westwood RF, et al. Further evidence to support the melanocytic origin of MDA-MB-435. Mol Pathol 2002;55:294–9.
    OpenUrlAbstract/FREE Full Text
  54. ↵
    Hahm HA, Dunn VR, Butash KA, et al. Combination of standard cytotoxic agents with polyamine analogues in the treatment of breast cancer cell lines. Clin Cancer Res 2001;7:391–9.
    OpenUrlAbstract/FREE Full Text
  55. ↵
    Li CJ, Li YZ, Pinto AV, et al. Potent inhibition of tumor survival in vivo by β-lapachone plus taxol: combining drugs imposes different artificial checkpoints. Proc Natl Acad Sci 1999;96:13369–74.
    OpenUrlAbstract/FREE Full Text
  56. ↵
    Bergeron RJ, Neims AH, McManis JS, et al. Synthetic polyamine analogues as antineoplastics. J Med Chem 1998;31:1183–90.
    OpenUrl
  57. ↵
    Casero RA Jr, Celano P, Ervin SJ, et al. High specific induction of spermidine/spermine N1-acetyltransferase in a human large cell lung carcinoma. Biochem J 1990;270:615–20.
    OpenUrlAbstract/FREE Full Text
  58. ↵
    Seely JE, Pegg AE. Ornithine decarboxylase (mouse kidney). Methods Enzymol 1983;94:158–61.
    OpenUrlCrossRefPubMed
  59. ↵
    Bradford MM. A rapid and sensitive methods for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54.
    OpenUrlCrossRefPubMed
PreviousNext
Back to top
Molecular Cancer Research: 2 (2)
February 2004
Volume 2, Issue 2
  • Table of Contents
  • About the Cover

Sign up for alerts

View this article with LENS

Open full page PDF
Article Alerts
Sign In to Email Alerts with your Email Address
Email Article

Thank you for sharing this Molecular Cancer Research article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Regulation of Polyamine Analogue Cytotoxicity by c-Jun in Human MDA-MB-435 Cancer Cells1 1 NIH grants P50CA88843 (N. E. D.) and CA51085 (R. A. C.), Breast Cancer Research Foundation grant (N. E. D.), and DOD grant DAMD 17-03-1-0376 (Y. H.).
(Your Name) has forwarded a page to you from Molecular Cancer Research
(Your Name) thought you would be interested in this article in Molecular Cancer Research.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
Regulation of Polyamine Analogue Cytotoxicity by c-Jun in Human MDA-MB-435 Cancer Cells1 1 NIH grants P50CA88843 (N. E. D.) and CA51085 (R. A. C.), Breast Cancer Research Foundation grant (N. E. D.), and DOD grant DAMD 17-03-1-0376 (Y. H.).
Yi Huang, Judith C. Keen, Erin Hager, Renee Smith, Amy Hacker, Benjamin Frydman, Aldonia L. Valasinas, Venodhar K. Reddy, Laurence J. Marton, Robert A. Casero Jr. and Nancy E. Davidson
Mol Cancer Res February 1 2004 (2) (2) 81-88;

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Share
Regulation of Polyamine Analogue Cytotoxicity by c-Jun in Human MDA-MB-435 Cancer Cells1 1 NIH grants P50CA88843 (N. E. D.) and CA51085 (R. A. C.), Breast Cancer Research Foundation grant (N. E. D.), and DOD grant DAMD 17-03-1-0376 (Y. H.).
Yi Huang, Judith C. Keen, Erin Hager, Renee Smith, Amy Hacker, Benjamin Frydman, Aldonia L. Valasinas, Venodhar K. Reddy, Laurence J. Marton, Robert A. Casero Jr. and Nancy E. Davidson
Mol Cancer Res February 1 2004 (2) (2) 81-88;
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • Introduction
    • Results
    • Discussion
    • Materials and Methods
    • Acknowledgments
    • Footnotes
    • References
  • Figures & Data
  • Info & Metrics
  • PDF
Advertisement

Related Articles

Cited By...

More in this TOC Section

  • GRM1 Suppression in Human Melanoma Cells
  • SINE Retrotransposons Cause Epigenetic Reprogramming
  • Genomic Targets and Role in Cell Survival of AEBP1
Show more Cancer Genes and Genomics
  • Home
  • Alerts
  • Feedback
  • Privacy Policy
Facebook  Twitter  LinkedIn  YouTube  RSS

Articles

  • Online First
  • Current Issue
  • Past Issues
  • Rapid Impact Archive
  • Meeting Abstracts

Information for

  • Authors
  • Subscribers
  • Advertisers
  • Librarians

About MCR

  • About the Journal
  • Editorial Board
  • Permissions
  • Submit a Manuscript
AACR logo

Copyright © 2021 by the American Association for Cancer Research.

Molecular Cancer Research
eISSN: 1557-3125
ISSN: 1541-7786

Advertisement