Competency-based assessment for the training of PhD students and early-career scientists.

The training of PhD students and early-career scientists is largely an apprenticeship in which the trainee associates with an expert to become an independent scientist. But when is a PhD student ready to graduate, a postdoctoral scholar ready for an independent position, or an early-career scientist ready for advanced responsibilities? Research training by apprenticeship does not uniformly include a framework to assess if the trainee is equipped with the complex knowledge, skills and attitudes required to be a successful scientist in the 21st century. To address this problem, we propose competency-based assessment throughout the continuum of training to evaluate more objectively the development of PhD students and early-career scientists.

Targeted overexpression of OGFr in epithelium of transgenic mice suppresses cell proliferation and impairs full-thickness wound closure.

The opioid growth factor (OGF) and its receptor, OGFr, play a regulatory role in cell proliferation, and maintain homeostasis through a tonically active negative feedback mechanism. To directly evaluate the repercussion of increased OGFr expression and consequent gain-of-function in epithelium, bovine keratin 5 promoter elements were used to direct the expression of OGFr to skin in a tetracycline-regulated manner. Three founder lines overexpressing OGFr (OGFrTG/K5-tTA) were established. Evidence for increased OGFr in the epithelium included a three-fold increase in OGFr binding activity, as well as significant increases in OGFr protein, as monitored by semiquantitative immunohistochemistry. DNA synthesis in target epithelium, including cornea, tongue, and skin of transgenic mice was decreased 41% to 80% from wild-type littermates; the liver, a nonepithelial organ, was not altered. Decreased DNA synthesis in corneal epithelium induced by transgenic expression of OGFr was further reduced by treatment with exogenous OGF but reversed by exposure to the opioid antagonist, naloxone. The number of cell layers in both epidermis and cornea of OGFrTG/K5-tTA animals was reduced nearly 45% from wild-type mice. Full-thickness wounds in mice overexpressing OGFr healed 37% to 75% slower than wild-type littermates. These data demonstrate for the first time that stable genetic amplification of OGFr downregulates homeostatic cell proliferation, as well as pathophysiological processes with respect to wound repair. These mice also can serve as a valuable model to dissect the mechanism of OGF-OGFr action and may be important in understanding the etiology, pathogenesis, and treatment of epithelium-related diseases.

Effects of fish oil and Tamoxifen on preneoplastic lesion development and biomarkers of oxidative stress in the early stages of N-methyl-N-nitrosourea-induced rat mammary carcinogenesis.

Epidemiologic studies on the protective role of omega-3 fatty acids (n:3) on breast cancer prevention remain inconclusive but studies in preclinical models provide more positive outcome. However, the mechanisms accounting for the protective effect of n:3 are not defined. In the present study, conducted in the N-methyl-N-nitrosourea-induced rat mammary carcinogenesis model, we examined the effects of n:3 individually and in combination with the anti-estrogen Tamoxifen (Tam) on a comprehensive panel of systemic and preneoplastic mammary gland restricted biomarkers which may be critical in the progression to invasive cancer. We observed that fish oil (FO) rich diets significantly reduced Ki67 expression in hyperplastic lesions, while cleaved caspase-3 expression was not affected. Dietary FO and/or Tam did not have major effects on systemic oxidative stress biomarkers, based on oxidative damage to DNA measured as 8-hydroxy-2-deoxyguanosine (8-OH-dG) and lipid peroxidation assessed as thiobarbituric acid reactive substances (TBARS). Tissue levels of 8-isoprostane, on the other hand, were markedly reduced (p<0.0001) in FO-fed rats, possibly as a result of FO-induced depletion of arachidonic acid in the mammary gland. These results suggest that the protective effect of n:3 in this experimental system is not mediated by changes in the levels of oxidative stress but may result from suppression of arachidonic acid-specific pathways.

The effects of Tamoxifen and fish oil on mammary carcinogenesis in polyoma middle T transgenic mice.

In these experiments, we tested the hypothesis that inhibition of the estrogen receptor (ER) with Tamoxifen and activation of PPARγ with fish oil (FO) rich in omega-3 (n-3; known PPAR agonists) inhibit the development of hormone-independent breast cancer in view of the known crosstalk between the ER and PPARγ pathways. We selected the polyoma middle T transgenic mouse model, since in this system the development of ER- tumors is preceded by ER positive preneoplastic lesions. Tamoxifen admixed with a 20% corn oil (CO) modified AIN-76A diet delayed mammary carcinogenesis and inhibited tumor multiplicity, volume, and weight in a dose-dependent (1, 10, and 100 ppm) fashion. Administration of increasing concentrations of FO in the diet (5%, 10%, and 17%) did not affect any of the tumor parameters. Combined administration of different doses of Tamoxifen and FO delayed carcinogenesis and suppressed tumor multiplicity and volume to the same extent as Tamoxifen alone. Mice fed 10% FO exhibited the expected increase in n-3/n-6 ratio in plasma and tumor based on diet analysis. Further increase in the n-3/n-6 ratio was not observed in mice fed the 17% FO diet. FO reduced tissue levels of arachidonic acid and its metabolite PGF-2α. Our results support the role of ER expression by preneoplastic lesions in the development of hormone-independent tumors and consequently the importance of including ER targeting in combination with mechanistically based novel chemopreventive agents.

The impact of fish oil on the chemopreventive efficacy of tamoxifen against development of N-methyl-N-nitrosourea-induced rat mammary carcinogenesis.

The antiestrogen tamoxifen reduces breast cancer incidence in high-risk women but is unable to inhibit the development of hormone-independent tumors. Omega-3 polyunsaturated fatty acids (n-3 PUFA), known ligands of the peroxisome proliferator activated receptor-gamma (PPARgamma), generally exert tumor-suppressive effects. Based on the known crosstalk between the estrogen and the PPARgamma receptors, we tested the hypothesis that the combination of tamoxifen with n-3 PUFA results in a superior antitumor action over the individual interventions. In this study, we report for the first time that the combination of a fish oil diet rich in n-3 PUFA and tamoxifen seemed to inhibit N-methyl-N-nitrosourea-induced mammary carcinogenesis, tumor multiplicity, and volume to a greater extent than the individual interventions. The potential superiority of the combination was particularly evident at a suboptimal dose of tamoxifen, which, by itself, was unable to significantly decrease tumor development. Because activation of PPARgamma is known to inhibit oxidative stress, we examined the effects of our interventions on circulating and tumor levels of glutathione, a major intracellular antioxidant. Our results indicate that reduction in the level of oxidative stress may be a potential mechanism by which the n-3 PUFA-rich diet potentiated the tumor-suppressive effect of tamoxifen. Our interventions were well tolerated without evidence of toxicity. Combined administration of tamoxifen and n-3 PUFA is a promising new approach to breast cancer prevention. Because of its safety, this combination can quickly be translated to the clinic if its superiority can be supported by future studies.

Dependence on nuclear localization signals of the opioid growth factor receptor in the regulation of cell proliferation.

The opioid growth factor receptor (OGFr) mediates the inhibitory action of OGF on cell replication of normal and neoplastic cells. The spatiotemporal course of OGFr nucleocytoplasmic trafficking was determined with a probe of full-length OGFr fused to enhanced green fluorescent protein (eGFP). Translation of OGFr required 8.5 hours, and transit into the nucleus required 8 hours; OGFr remained in the nucleus for 8 days. OGFr was initially expressed on the outer nuclear envelope, transited to the paranuclear cytoplasm, and into the nucleus. Transport through the nuclear pore was elucidated by mutation of the nuclear localization signal (NLS) sequences in full-length OGFr. Mutation of each NLS reduced nuclear localization by 5%-50%, whereas simultaneous mutation of NLS383-386 and NLS456-460 abolished OGFr-eGFP nuclear localization in 80% of the cells. To determine whether intact NLSs are important for the inhibition of cell proliferation, DNA synthesis was monitored with BrdU. Wild-type OGFr-eGFP-transfected cells had 20% BrdU-positive cells, whereas cells with simultaneous mutation of all three NLS sites had a 70% labeling index. These results indicate that the regulation of cell proliferation by the OGF-OGFr axis is dependent on nucleocytoplasmic translocation and reliant on the integrity of two NLSs in OGFr to interact with transport receptors.

The OGF-OGFr axis utilizes the p16INK4a and p21WAF1/CIP1 pathways to restrict normal cell proliferation.

Opioid growth factor (OGF) is an endogenous opioid peptide ([Met(5)]enkephalin) that interacts with the OGF receptor (OGFr) and serves as a tonically active negative growth factor in cell proliferation of normal cells. To clarify the mechanism by which OGF inhibits cell replication in normal cells, we investigated the effect of the OGF-OGFr axis on cell cycle activity in human umbilical vein endothelial cells (HUVECs) and human epidermal keratinocytes (NHEKs). OGF markedly depressed cell proliferation of both cell lines by up to 40% of sterile water controls. Peptide treatment induced cyclin-dependent kinase inhibitor (CKI) p16(INK4a) protein expression and p21(WAF1/CIP1) protein expression in HUVECs and NHEKs, but had no effect on p15, p18, p19, or p27 protein expression in either cell type. Inhibition of either p16(INK4a) or p21(WAF1/CIP1) activation by specific siRNAs blocked OGF inhibitory action. Human dermal fibroblasts and mesenchymal stem cells also showed a similar dependence of OGF action on p16(INK4a) and p21(WAF1/CIP1). Collectively, these results indicate that both p16(INK4a) and p21(WAF1/CIP1) are required for the OGF-OGFr axis to inhibit cell proliferation in normal cells.

The OGF-OGFr axis utilizes the p21 pathway to restrict progression of human pancreatic cancer.

BACKGROUND: Pancreatic cancer is the 4th leading cause of death from cancer in the U.S. The opioid growth factor (OGF; [Met5]-enkephalin) and the OGF receptor form an inhibitory growth regulatory system involved in the pathogenesis and treatment of pancreatic cancer. The OGF-OGFr axis influences the G0/G1 phase of the cell cycle. In this investigation, we elucidate the pathway of OGF in the cell cycle. RESULTS: Using BxPC-3 cells, OGF decreased phosphorylation of retinoblastoma (Rb) protein without changing total Rb. This change was correlated with reduced cyclin-dependent kinase protein (Cdk) 2 kinase activity, but not total Cdk2. OGF treatment increased cyclin-dependent kinase inhibitor (CKI) p21 protein expression in comparison to controls, as well levels of p21 complexed with Cdk2. Naloxone abolished the increased expression of p21 protein by OGF, suggesting a receptor-mediated activity. p21 specific siRNAs blocked OGF's repressive action on proliferation in BxPC-3, PANC-1, and Capan-2 cells; cells transfected with negative control siRNA had no alteration in p21 expression, and therefore were inhibited by OGF. CONCLUSION: These data are the first to reveal that the target of cell proliferative inhibitory action of OGF in human pancreatic cancer is a p21 CKI pathway, expanding strategies for diagnosis and treatment of these neoplasias.

Role of non-receptor and receptor tyrosine kinases (TKs) in the antitumor action of alpha-difluoromethylornithine (DFMO) in breast cancer cells.

We have shown that administration of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine (PA) biosynthesis, reduces the invasive and metastatic properties of MDA-MB-435 breast cancer cells while activating multiple signal transduction pathways, including MAPK, Stat3, Stat1, and JNK. Since the activity of these signaling mechanisms is frequently regulated by upstream tyrosine kinases (TKs), we tested whether non-receptor and receptor TKs may be involved in the signaling and biological effects of DFMO in MDA-MB-435 cells. Treatment with DFMO (1 mM for 48 h) did not affect Src phosphorylation (Tyr 416). Administration of the Src-family members inhibitor PP-1 (1 microM), blocked Src phosphorylation in the absence and in the presence of DFMO, but did not block the signaling effects of DFMO (increased phosphorylation of Stat3, Stat1, ERK and JNK). PP-1 treatment, on the other hand, inhibited the invasiveness of MDA-MB-435 cells in matrigel and potentiated the anti-invasive effect of DFMO. Next, we focused on the role of receptor TK. Western analysis of cell lysates from MDA-MB-435 cells failed to show the presence of EGF-R and HER-2neu but demonstrated the expression of c-Met, the receptor for hepatocyte growth factor (HGF). Therefore, we tested the effect of DFMO on the HGF/c-Met pathway which is strongly implicated in the progression of human breast cancer. We found that DFMO treatment blocked HGF-induced c-Met phosphorylation in MDA-MB-435 cells, suggesting that its anti-invasion action may be mediated, at least in part, by blocking c-Met signaling. Next, we showed that 1 mM DFMO suppressed HGF induced invasiveness of MDA-MB-435 cells in matrigel. Combination administration of DFMO with suboptimal doses of PHA-665752, a specific c-Met inhibitor, reduced invasiveness to an even greater extent than the individual treatment. These findings indicate that Src-family members, while not involved in DFMO action, promote invasiveness of breast cancer cells and their inhibition may enhance the antitumor effect of PA depletion. Our data also point to inhibition of HGF/c-Met pathway as a possible novel approach to enhancing the antitumor action of DFMO.

Activation of protein kinase A (PKA) signaling mitigates the antiproliferative and antiinvasive effects of alpha-difluoromethylornithine in breast cancer cells.

We have shown that alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, the first and rate-limiting enzyme in polyamine synthesis, has significant antiproliferative and antiinvasive effects in breast cancer cells. We have also reported that these antitumor effects are associated with activation of multiple signaling pathways, including STAT-3, STAT-1, Jun-N-Terminal kinase (JNK), and Mitogen activated protein kinase (MAPK), the latter being found to mediate its antiinvasive action in MDA-MB-435 cells. The present experiments were designed to test the effect of DFMO on the protein kinase A (PKA) pathway and determine its biological significance. We found that DFMO administration (1 mM) to MDA-MB-435 breast cancer cells significantly increased cAMP response element (CRE)-binding protein (CREB) phosphorylation as well as the transactivation of pCRE-luc, a CREB-dependent promoter activated by PKA. To determine the significance of this biochemical effect of DFMO, we used the PKA inhibitor H89 which, as expected, suppressed in a dose-dependent manner (1 and 10 microM) basal and DFMO-induced CREB phosphorylation in our system. Administration of H89 alone was able to suppress proliferation of MDA-MB-435 cells when used at a concentration (10 microM) shown to completely block basal CREB phosphorylation. At concentrations of 0.5 and 1 muM, H89 treatment, while having no antiproliferative effect of its own, potentiated in a dose-dependent fashion the growth inhibitory action of a suboptimal concentration of DFMO (0.01 mM). Ten micromoles of H89 reduced invasiveness of MDA-MB-435 cells in matrigel by approximately 40% (an effect similar to that of 1 mM DFMO). The combination treatment further reduced invasiveness by approximately 80% (P < 0.01 versus the individual treatments). H89 treatment (10 microM) partially reduced DFMO-induced phosphorylation of STAT-3 but not that of STAT-1, Extracellular regulated kinase (ERK), and JNK. In conclusion, our results indicate that PKA signaling exerts proproliferative and proinvasive effects in our experimental system. Therefore, its activation by DFMO represents a compensatory mechanism which should be blocked in order to maximize the antitumor action of the drug. Our data are also consistent with the notion that STAT-3 activation by DFMO is at least in part mediated through the PKA pathway.

Effects of polyamine depletion by alpha-difluoromethylornithine on in vitro and in vivo biological properties of 4T1 murine mammary cancer cells.

Increased polyamine synthesis has been associated with proliferation and progression of breast cancer, and thus, is a potential target for anticancer therapy. Polyamine depletion by alpha-difluoromethylornithine (DFMO) has been shown to decrease pulmonary and bone metastasis from human breast cancer cell xenografts. Following these observations, this study was designed to test the effects of DFMO on in vitro and in vivo features of the highly invasive and metastatic 4T1 murine mammary cancer cells. DFMO inhibited proliferation, caused G1-S arrest, and suppressed in vitro invasiveness of 4T1 cells. In contrast to our previous findings with MDA-MB-435 cells, DFMO did not affect the activation of signal transducers and activator of transcription 3, c-Jun N-terminal kinase, and extracellular signal-regulated kinase, but decreased phosphorylation of p38. DFMO did not alter the expression of Twist. DFMO delayed the orthotopic growth of 4T1 xenografts in association with suppressed putrescine and spermidine levels but increased levels of spermine. DFMO did not affect pulmonary metastasis when primary tumors of control and DFMO-treated mice were matched for size. Interestingly, DFMO reduced Ki-67 expression only in the primary tumors but did not affect its expression in the metastatic tumors in the lung. Cleaved caspase-3 expression was not affected by DFMO in either the primary tumors or the pulmonary metastasis. In summary, DFMO treatment markedly inhibited in vitro proliferation and invasiveness of 4T1 cells and retarded the growth of orthotopic xenografts in mice. The failure of DFMO to inhibit pulmonary metastasis in this system appears to be due, at least in part, to its lack of antiproliferative effect at the metastatic sites.

The opioid growth factor (OGF)-OGF receptor axis uses the p16 pathway to inhibit head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) represents 5.5% of malignancies worldwide, with approximately 30,000 new cases and approximately 11,000 deaths reported in the United States annually. The opioid growth factor (OGF; [Met(5)]-enkephalin) and the OGF receptor (OGFr) form an endogenous growth regulating system; the OGF-OGFr axis influences the G(0)-G(1) phase of the cell cycle in HNSCC. Cells treated with small interfering RNA (siRNA) for OGFr no longer responded to the growth inhibitory effects of OGF or the growth stimulatory effects of naltrexone, indicating that these activities are entirely mediated by OGFr. In this investigation, we examined the precise target of OGF in the cell cycle. Using SCC-1 cells, OGF decreased the phosphorylation of retinoblastoma protein. This change was correlated with reduced Cdk4, but not Cdk2, kinase activity. OGF treatment increased cyclin-dependent kinase inhibitor p16 protein expression. Importantly, p16 complexed with Cdk4 was increased by OGF treatment at all time points, consistent with the hypothesis that OGF mediated growth inhibition through p16. Blockade of OGF-OGFr interactions with naloxone abolished the increased expression of p16 protein by OGF. Inhibition of p16 (INK4a) activation by p16-specific siRNA blocked OGF's repressive action on proliferation of SCC-1, CAL-27, and SCC-4 HNSCC cells. These data are the first to reveal that the target of cell proliferative inhibitory action of OGF in human HNSCC is a cyclin-dependent kinase inhibitory pathway, and this may be useful in the diagnosis and treatment of HNSCC.

Effects of alpha-difluoromethylornithine on thrombospondin-1 production by human breast cancer cells.

We have previously observed that inhibition of polyamine biosynthesis with alpha-difluoromethylornithine (DFMO) upregulates production of thrombospondin-1 (TSP-1), an extracellular matrix protein with potent anti-angiogenic and antimetastatic properties, by MDA-MB-435 human breast cancer cells in culture. The present experiments were designed to investigate the mechanisms by which DFMO regulates TSP-1 production in this system. 35S-methionine pulse chase experiments indicated that DFMO administration increased TSP-1 synthesis by approximately 6-fold, while it slightly but significantly decreased protein half-life from 35 to 28 min. DFMO treatment increased steady state TSP-1 mRNA levels by 2-fold in MDA-MB-435 cells. TSP-1 promoter reporter studies indicated that this increase was largely due to activation of transcription. Analysis of distribution of TSP-1 mRNA levels between non-polysomal, subpolysomal and polysomal fractions in control and DFMO-treated cells suggested a major stimulatory effect of the drug on TSP-1 translation. A similar increase in TSP-1 transcription and translation in response to DFMO treatment was also observed in vivo in MDA-MB-435 breast cancer xenografts. Surprisingly however, we failed to detect an increase in TSP-1 protein as assessed by Western blot analysis. The reason for this unexpected finding is unknown but may be due to DFMO-induced stimulation of TSP-1 secretion into the systemic circulation, thus preventing its accumulation within the tumor.

The TATA binding protein associated factor 4b (TAF4b) mediates FSH stimulation of the IGFBP-3 promoter in cultured porcine ovarian granulosa cells.

We have established the gene for IGF binding protein-3 (IGFBP-3) as a target for FSH action. FSH effects on this gene require the PKA pathway as well as the PI-3 kinase and MAPK pathways. At the IGFBP-3 promoter, FSH effects depend on a site for TATA box binding protein (TBP) and formation of a high molecular weight transcription complex. To further elucidate FSH effects on the downstream events involving the TBP site, we cloned a pig TAF4b cDNA into a P-Flag expression vector. By co-transfecting granulosa cells with the IGFBP-3 promoter, we found that TAF4b mimics and enhances FSH induction of IGFBP-3 reporter activity. Using RT-PCR we showed that FSH stimulates expression of TAF4b. This would suggest that the role of TAF4b in follicular development is regulated by FSH. TAF4b may thus be the TFIID component that binds to the TBP site on the IGFBP-3 promoter and is essential for FSH induction of IGFBP-3.

Overexpression of the opioid growth factor receptor potentiates growth inhibition in human pancreatic cancer cells.

The opioid growth factor (OGF), [Met5]-enkephalin, and OGF receptor (OGFr) form an inhibitory axis regulating the growth of human pancreatic cancer. This study examined whether overexpression of OGFr decreases the growth of pancreatic cells in vitro. MIA PaCa-2 cells were transfected with OGFr cDNA, and six clonal lines were examined for protein expression and function. OGFr binding assays revealed a 2.3- to 5.6-fold increase in binding capacity from wild-type (WT) and empty vector (EV) controls; binding affinity was comparable in all groups. OGFr protein expression, as measured by immunohistochemistry and Western blotting, was enhanced in clonal cell lines compared to controls. Doubling times of OGFr clonal lines were 47-91% longer than in the WT/EV groups for all but one clonal line. DNA synthesis of cells overexpressing OGFr was diminished from the WT/EV groups by 28-52%. Addition of exogenous OGF further reduced (14-31%) the cell growth of clonal lines, and the effects of exogenous OGF were receptor-mediated. Exposure of cells overexpressing OGFr to naltrexone increased the cell number by up to 9.4-fold. OGF was identified as the only opioid peptide to depress cell replication in the transfected cell lines. Neutralization of endogenous OGF with antibodies to this peptide elevated the cell number in clonal cell lines. These data identify OGFr at the molecular level as integral to regulating the cell replication of human pancreatic cancer, and support treatment modalities that amplify OGFr in order to decrease the growth of these neoplasias.

Overexpression of the opioid growth factor receptor downregulates cell proliferation of human squamous carcinoma cells of the head and neck.

The opioid growth factor (OGF) is a constitutively expressed negative growth regulator whose action is mediated by the OGF receptor (OGFr). The OGF-OGFr axis tonically regulates the growth of human squamous cell carcinoma of the head and neck (SCCHN). To examine the repercussions of amplifying OGFr in SCCHN, constructs were prepared to overexpress OGFr in SCC-1 cells; six clonal lines were examined. OGFr binding assays of clonal cells revealed a 2.4- to 8.4-fold increase in binding capacity compared to wild-type (WT) and empty vector (EV) controls; binding affinity was comparable in all groups. OGFr protein expression, as measured by quantitative immunohistochemistry and Western blotting, was increased in clonal cell lines compared to controls. Under standard growth conditions the cell number of the OGFr clonal lines was reduced by 11 to 68% from the WT group, and doubling times were 7 to 67% longer. Addition of exogenous OGF further reduced (8 to 37%) cell growth of the clonal lines. Depletion of endogenous OGF with antibodies to this peptide increased growth 2-fold in cells amplifying OGFr relative to increases of 32 and 34% for the WT and EV groups, respectively. DNA synthesis of cells overexpressing OGFr was reduced from the WT group by 46 to 75%. These data indicate that the OGF receptor is integral to cell replication of SCCHN, and support treatment modalities that amplify OGFr in order to decrease the growth of these neoplasias.

Effects of polyamine depletion by alpha-difluoromethylornithine on in vitro and in vivo biological properties of 4T1 murine mammary cancer cells.

Increased polyamine synthesis has been associated with proliferation and progression of breast cancer, and thus, is a potential target for anti-cancer therapy. Polyamine depletion by DFMO has been shown to decrease pulmonary and bone metastasis from human breast cancer cell xenografts. Following these observations, this study was designed to test the effects of DFMO on in vitro and in vivo features of the highly invasive and metastatic 4T1 murine mammary cancer cells. DFMO inhibited proliferation, caused G1-S arrest, and suppressed in vitro invasiveness of 4T1 cells. In contrast to our previous findings with MDA-MB-435 cells, DFMO did not affect the activation of STAT3, JNK, and ERK, but decreased phosphorylation of p38. DFMO did not alter the expression of Twist. DFMO delayed the orthotopic growth of 4T1 xenografts in association with suppressed putrescine and spermidine levels but increased levels of spermine. DFMO did not affect pulmonary metastasis when primary tumors of control and DFMO-treated mice were matched for size. Interestingly, DFMO reduced Ki-67 expression only in the primary tumors but did not affect its expression in the metastatic tumors in the lung. Cleaved caspase-3 expression was not affected by DFMO in either the primary tumors or pulmonary metastasis. In summary, DFMO treatment markedly inhibited in vitro proliferation and invasiveness of 4T1 cells and retarded the growth of orthotopic xenografts in mice. The failure of DFMO to inhibit pulmonary metastasis in this system appears to be due, at least in part, to its lack of anti-proliferative effect at the metastatic sites.

Interaction between Polyamines and the Mitogen-Activated Protein Kinase Pathway in the Regulation of Cell Cycle Variables in Breast Cancer Cells.

Inhibition of polyamine biosynthesis with alpha-difluoromethylornithine (DFMO) has been shown to inhibit proliferation of breast cancer cells although its mechanism of action has not been fully elucidated. To address this issue, we tested the effects of DFMO on cell cycle variables of MDA-MB-435 human breast cancer cells in culture. We also focused on the possible mediatory role of the mitogen-activated protein kinase (MAPK) pathway on the cell cycle effects of DFMO because this compound has been shown to activate MAPK signaling. We found that DFMO caused a p53-independent increase in p21 and its association with cyclin-dependent kinase (cdk)-2 and decreased cdk-2 protein as well as its phosphorylation on Thr160. In addition, DFMO markedly suppressed the expression of the full-length and low molecular weight forms of cyclin E. These effects of DFMO were reversible with exogenous putrescine, thus indicating that they are specifically mediated through polyamine depletion. Cdk-2 activity was drastically reduced in DFMO-treated breast cancer cells which exhibited a reduction in retinoblastoma (Rb) phosphorylation and protein. As a predictable consequence of these effects, DFMO caused a G1-S block. In addition, DFMO inhibited G2-M transition, most likely as a result of its induction of p21 expression. Inhibition of the MAPK pathway with PD98059 or U0126 blocked the DFMO-induced induction of p21 and the reduction of cdk-2 protein. PD98059 reversed the G2-M block induced by DFMO (probably as a result of suppression of p21) but not the G1-S arrest. MDA-MB-435 cells treated with PD98059 or U0126 in the presence and absence of DFMO exhibited a marked increase in the expression of p27 and its association with cdk-2, a decrease in phosphorylation of cdk-2 on Thr160, and a decrease in cyclin E expression. As predicted, PD98059 treatment reduced cdk-2 activity and Rb phosphorylation while reversing the decrease in Rb protein induced by DFMO. Neither DFMO nor PD98059, either alone or in combination, reduced cdk-4 activity despite a marked induction in p15 expression caused by DFMO. Our results indicate that activation of the MAPK pathway accounts for some of the effects of DFMO on cell cycle events of breast cancer cells. Inhibition of the MAPK pathway, however, does not reverse the cell cycle arrest induced by DFMO because of activation of alternative mechanisms leading to suppression of cdk-2 activity.

Effects of polyamine synthesis inhibitors on primary tumor features and metastatic capacity of human breast cancer cells.

We have previously reported that inhibition of polyamine biosynthesis with alpha-difluoromethylornithine (DFMO) reduces pulmonary metastasis from MDA-MB-435 human breast cancer xenografts without affecting the volume of the primary tumors (Manni et al. Clin Exp Mets 20:321, 2003). In these experiments, we show that DFMO treatment (2% in drinking H(2)O) reduced the growth fraction of the primary tumors by 60%. However, this effect was counter-balanced by a similar reduction in non-apoptotic necrosis, thus accounting for the preservation of tumor volume in DFMO-treated mice. DFMO treatment caused a 4-fold increase in cytoplasmic staining for cleaved caspase-3 (as opposed to the nuclear staining observed in control tonsil tissue) in the absence of histologic evidence of apoptosis. DFMO treatment reduced the number of mice with pulmonary metastasis by approximately 80% and the number of metastasis per mouse by >90% in association with a reduction in invasiveness of the primary tumor in the surrounding dermis and muscle by approximately 30%. DFMO treatment increased ERK phosphorylation in the tumors, an effect that has been found by us in vitro to be causally linked to the anti-invasive effect of the drug (Manni et al. Clin Exp Metast 2004; 21: 461]. DFMO also increased tyrosine phosphorylation of STAT-3 and expression of STAT-1 and JNK proteins. Administration of SAM486A (1 mg/kg/i.p. daily), an inhibitor of S-adenosylmethionine decarboxylase, either individually or in combination with DFMO, was not found to exert any biological or biochemical effects, most likely as a result of its failure to suppress tissue polyamine levels under these experimental conditions.