Search

PDF file - 383K, Rapamycin does not sensitize keratinocytes to UVB-induced cell death

PDF file - 359K, Vehicle-treated K5-CreERT2;mTORfl/fl, 4OHT-treated mTORfl/fl, and vehicle-treated mTORfl/fl mice do not differ

UV radiation is the major risk factor for developing skin cancer, the most prevalent cancer worldwide. Several studies indicate that mTOR signaling is activated by UVB and may play an important role in skin tumorigenesis. mTOR exists in two functionally and compositionally distinct protein complexes: the rapamycin-sensitive mTOR complex 1 (mTORC1) and the rapamycin-resistant mTOR complex 2 (mTORC2). The purpose of these studies was to investigate the roles of the two mTOR complexes in UVB-mediated proliferation and apoptosis in the skin. We used rapamycin, a pharmacologic inhibitor of mTORC1, and an inducible mTOR-deficient (K5-CreERT2;mTORfl/fl) mouse model that allows epidermal-specific disruption of mTOR following topical treatment with 4-hydroxytamoxifen (4OHT). Rapamycin blocked UVB-induced phosphorylation of S6K, the downstream target of mTORC1, and significantly reduced UVB-stimulated epidermal proliferation and cell-cycle progression, but had no effect on cell death. In contrast, mTOR deletion, which attenuated UVB-induced phosphorylation of both S6K and the mTORC2 target AKTSer473, significantly increased apoptosis both in vivo and in keratinocyte cultures, in addition to reducing hyperproliferation following UVB irradiation. The role of mTORC2 in UVB-induced prosurvival signaling was verified in Rictor−/− mouse embryo fibroblasts, which lack functional mTORC2 and were more sensitive to UVB-induced apoptosis than controls. These studies show that mTORC1 and mTORC2 play unique but complementary roles in controlling proliferation and apoptosis in the skin. Our findings underscore the importance of both mTOR complexes in mediating UVB-induced signaling in keratinocytes and provide new insight into the pathogenesis of skin cancer. Cancer Prev Res; 5(12); 1394–404. ©2012 AACR.

PDF file - 76K

PDF file - 1MB, Rapamycin attenuates TPA-induced epidermal thickening

Supplementary Figure 4 from Ras Transformation of RIE-1 Cells Activates Cap-Independent Translation of Ornithine Decarboxylase: Regulation by the Raf/MEK/ERK and Phosphatidylinositol 3-Kinase Pathways

Ornithine decarboxylase (ODC) is the first and generally rate-limiting enzyme in polyamine biosynthesis. Deregulation of ODC is critical for oncogenic growth, and ODC is a target of Ras. These experiments examine translational regulation of ODC in RIE-1 cells, comparing untransformed cells with those transformed by an activated Ras12V mutant. Analysis of the ODC 5′ untranslated region (5′UTR) revealed four splice variants with the presence or absence of two intronic sequences. All four 5′UTR species were found in both cell lines; however, variants containing intronic sequences were more abundant in Ras-transformed cells. All splice variants support internal ribosome entry site (IRES)–mediated translation, and IRES activity is markedly elevated in cells transformed by Ras. Inhibition of Ras effector targets indicated that the ODC IRES element is regulated by the phosphorylation status of the translation factor eIF4E. Dephosphorylation of eIF4E by inhibition of mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) or the eIF4E kinase Mnk1/2 increases ODC IRES activity in both cell lines. When both the Raf/MEK/ERK and phosphatidylinositol 3-kinase/mammalian target of rapamycin pathways are inhibited in normal cells, ODC IRES activity is very low and cells arrest in G1. When these pathways are inhibited in Ras-transformed cells, cell cycle arrest does not occur and ODC IRES activity increases, helping to maintain high ODC activity. [Cancer Res 2007;67(10):4834–42]

Supplementary Figure 3 from Ras Transformation of RIE-1 Cells Activates Cap-Independent Translation of Ornithine Decarboxylase: Regulation by the Raf/MEK/ERK and Phosphatidylinositol 3-Kinase Pathways

Supplementary Figure 2 from Ras Transformation of RIE-1 Cells Activates Cap-Independent Translation of Ornithine Decarboxylase: Regulation by the Raf/MEK/ERK and Phosphatidylinositol 3-Kinase Pathways

Supplementary Figure 1 from Ras Transformation of RIE-1 Cells Activates Cap-Independent Translation of Ornithine Decarboxylase: Regulation by the Raf/MEK/ERK and Phosphatidylinositol 3-Kinase Pathways

Supplementary Figure Legends 1-4 from Ras Transformation of RIE-1 Cells Activates Cap-Independent Translation of Ornithine Decarboxylase: Regulation by the Raf/MEK/ERK and Phosphatidylinositol 3-Kinase Pathways

SummaryHerpes simplex virus type 1 (HSV-1) causes ocular and orofacial infections, which are generally well controlled by the host and nonlethal. In rare cases, HSV-1 causes encephalitis, which leads to permanent brain injuries, memory loss or even death. Host factors protect the organism from viral infections by activating the immune response. However, the factors that confer neuroprotection during viral encephalitis are unknown. Here we show that mammalian target of rapamycin complex 2 (mTORC2) is essential for the host survival of ocular HSV-1 infections in vivo. We found that the loss of mTORC2 causes systemic HSV-1 infection not only because of weak innate and adaptive immune responses but also due to increased ocular and neuronal cell death, which becomes lethal over time. Furthermore, we found that mTORC2 mediates cell survival channels through the inactivation of the proapoptotic factor FoxO3a. Our results demonstrate how mTORC2 potentiates host defenses against viral infections as well as implicating mTORC2 as a necessary host factor for survival. We anticipate our findings may help develop new therapeutic window for severe HSV-1 infections, such as herpes simplex encephalitis.

Non-melanoma skin cancer (NMSC) is the most commonly diagnosed cancer in the United States. Ultraviolet-B (UVB) irradiation is the primary carcinogen responsible for stimulating NMSC development. Ornithine Decarboxylase (ODC), the first rate-limiting enzyme in the synthesis of polyamines, is upregulated in response to a variety of proliferation stimuli, including UVB exposure. Our previous studies have demonstrated regulation of ODC synthesis by the mammalian target of rapamycin complex 1 (mTORC1) in cells transformed by oncogenic Ras. The goal of these studies was to better understand the link between mTORC1 and ODC in nontransformed cells treated with UVB. We show that the ablation of mTORC1 activity by conditional knockout of its essential component Raptor led to decreased levels of ODC protein both before and after exposure to 10 mJ/cm(2) UVB. Moreover, ODC mRNA was destabilized in the absence of Raptor, suggesting post-transcriptional regulation. We have previously shown that the ODC transcript is stabilized by the RNA binding protein (RBP) human antigen R (HuR), and the intracellular localization of HuR responds to changes in mTORC1 activity. To expand these studies, we investigated whether HuR functions to regulate ODC mRNA stability after UVB exposure. Our results show an increased localization of HuR to the cytoplasm after UVB exposure in wild-type cells compared to Raptor knockout cells, and this is accompanied by greater association of HuR with the ODC transcript. These data suggest that the localization of HuR in response to UVB is influenced, at least in part, by mTORC1 and that HuR can bind to and stabilize ODC mRNA after UVB exposure in an mTORC1-dependent manner.

INTRODUCTION AND OBJECTIVES:Advanced bladder cancer exhibits a unique degree of morphologic and molecular intratumoral heterogeneity. While tumor heterogeneity in bladder cancer is associated with ...

Nonmelanoma skin cancer (NMSC) is the most common cancer worldwide and the incidence continues to rise, in part due to increasing numbers in high-risk groups such as organ transplant recipients and those taking photosensitizing medications. The most significant risk factor for NMSC is ultraviolet radiation (UVR) from sunlight, specifically UVB, which is the leading cause of DNA damage, photoaging, and malignant transformation in the skin. Activation of apoptosis following UVR exposure allows the elimination of irreversibly damaged cells that may harbor oncogenic mutations. However, UVR also activates signaling cascades that promote the survival of these potentially cancerous cells, resulting in tumor initiation. Thus, the UVR-induced stress response in the skin is multifaceted and requires coordinated activation of numerous pathways controlling DNA damage repair, inflammation, and kinase-mediated signal transduction that lead to either cell survival or cell death. This review focuses on the central signaling mechanisms that respond to UVR and the subsequent cellular changes. Given the prevalence of NMSC and the resulting health care burden, many of these pathways provide promising targets for continued study aimed at both chemoprevention and chemotherapy.

Exposure to ultraviolet-B (UVB) irradiation, the principal cause of non-melanoma skin cancer (NMSC), activates both the rapamycin-sensitive mammalian target of rapamycin complex 1 (mTORC1) and the rapamycin-resistant mTORC2. We have previously reported that UVB-induced keratinocyte survival is dependent on mTORC2, though the specific mechanism is not well understood. FOXO3a is an important transcription factor involved in regulating cell survival. The activity of FOXO3a is reduced as a result of protein kinase B (AKT/PKB) activation, which is downstream of mTORC2; however, the specific function of FOXO3a during UVB-induced apoptosis is unclear. In this study, we establish that in cells with wild-type mTORC2 activity, FOXO3a is quickly phosphorylated in response to UVB and sequestered in the cytoplasm. In contrast, loss of mTORC2 causes FOXO3a to be localized to the nucleus and sensitizes cells to UVB-induced apoptosis. Furthermore, this sensitization is rescued by knockdown of FOXO3a. Taken together, these studies provide strong evidence that inhibition of mTORC2 enhances UVB-induced apoptosis in a FOXO3a-dependent manner, and suggest that FOXO3a activation by mTORC2 inhibitors may be a valuable chemopreventive target in NMSC.

Ornithine decarboxylase (ODC) is the first and usually rate-limiting enzyme in the polyamine biosynthetic pathway. In a normal physiological state, ODC is tightly regulated. However, during neoplastic transformation, ODC expression becomes upregulated. The studies described here show that the ODC mRNA transcript is destabilized by the RNA-binding protein tristetraprolin (TTP). We show that TTP is able to bind to the ODC mRNA transcript in both non-transformed RIE-1 cells and transformed Ras12V cells. Moreover, using mouse embryonic fibroblast cell lines that are devoid of a functional TTP protein, we demonstrate that in the absence of TTP both ODC mRNA stability and ODC enzyme activity increase when compared to wild-type cells. Finally, we show that the ODC 3' untranslated region contains cis acting destabilizing elements that are affected by, but not solely dependent on, TTP expression. Together, these data support the hypothesis that TTP plays a role in the post-transcriptional regulation of the ODC mRNA transcript.

Intratumoral heterogeneity in bladder cancer is a barrier to accurate molecular sub-classification and treatment efficacy. However, individual cellular and mechanistic contributions to tumor heterogeneity are controversial. We examined potential mechanisms of FOXA1 and PTEN inactivation in bladder cancer and their contribution to tumor heterogeneity. These analyses were complemented with inactivation of FOXA1 and PTEN in intermediate and luminal mouse urothelium. We show inactivation and reduced expression of FOXA1 and PTEN is prevalent in human disease, where PTEN and FOXA1 are downregulated by allelic loss and site-specific DNA hypermethylation, respectively. Conditional inactivation of both Foxa1 and Pten in intermediate/luminal cells in mice results in development of bladder cancer exhibiting squamous features as well as enhanced sensitivity to a bladder-specific carcinogen. In addition, FOXA1 is hypermethylated in basal bladder cancer cell lines, and this is reversed by treatment with DNA methyltransferase inhibitors. By integrating human correlative and in vivo studies, we define a critical role for PTEN loss and epigenetic silencing of FOXA1 in heterogeneous human disease and show genetic targeting of luminal/intermediate cells in mice drives squamous differentiation.

Nonmelanoma skin cancer (NMSC) is a major health concern worldwide. With increasing numbers in high-risk groups such as organ transplant recipients and patients taking photosensitizing medications, the incidence of NMSC continues to rise. Mouse models of NMSC allow us to better understand the molecular signaling cascades involved in skin tumor development in order to identify novel therapeutic strategies. Here we review the models designed to determine the role of the polyamines in NMSC development and maintenance. Elevated polyamines are absolutely required for tumor growth, and dysregulation of their biosynthetic and catabolic enzymes has been observed in NMSC. Studies using mice with genetic alterations in epidermal polyamines suggest that they play key roles in tumor promotion and epithelial cell survival pathways, and recent clinical trials indicate that pharmacological inhibitors of polyamine metabolism show promise in individuals at high risk for NMSC.

Activation of signaling dependent on the mammalian target of rapamycin (mTOR) has been demonstrated in a variety of human malignancies, and our previous work suggests that mTOR complex (mTORC) 1 and mTORC2 may play unique roles in skin tumorigenesis. The purpose of these studies was to investigate the function of mTORC2-dependent pathways in skin tumor development and the maintenance of established tumors. Using mice that allow spatial and temporal control of mTORC2 in epidermis by conditional knockout of its essential component Rictor, we studied the effect of mTORC2 loss on both epidermal proliferation and chemical carcinogenesis. The results demonstrate that mTORC2 is dispensable for both normal epidermal proliferation and the hyperproliferative response to treatment with tetradecanoyl phorbol acetate (TPA). In contrast, deletion of epidermal Rictor prior to initiation in DMBA/TPA chemical carcinogenesis was sufficient to dramatically delay tumor development and resulted in reduced tumor number and size compared with control groups. Silencing of Rictor expression in tumor-bearing animals triggered regression of established tumors and increased caspase-3 cleavage without changes in proliferation. In vitro experiments demonstrate an increased sensitivity to caspase-dependent apoptosis in the absence of rictor, which is dependent on mTORC2 signaling. These studies demonstrate that mTORC2 activation is essential for keratinocyte survival, and suggest that inhibition of mTORC2 has value in chemoprevention by eliminating carcinogen-damaged cells during the early stages of tumorigenesis, and in therapy of existing tumors by restricting critical pro-survival pathways.

UV radiation is the major risk factor for developing skin cancer, the most prevalent cancer worldwide. Several studies indicate that mTOR signaling is activated by UVB and may play an important role in skin tumorigenesis. mTOR exists in two functionally and compositionally distinct protein complexes: the rapamycin-sensitive mTOR complex 1 (mTORC1) and the rapamycin-resistant mTOR complex 2 (mTORC2). The purpose of these studies was to investigate the roles of the two mTOR complexes in UVB-mediated proliferation and apoptosis in the skin. We used rapamycin, a pharmacologic inhibitor of mTORC1, and an inducible mTOR-deficient (K5-CreERT2;mTORfl/fl) mouse model that allows epidermal-specific disruption of mTOR following topical treatment with 4-hydroxytamoxifen (4OHT). Rapamycin blocked UVB-induced phosphorylation of S6K, the downstream target of mTORC1, and significantly reduced UVB-stimulated epidermal proliferation and cell-cycle progression, but had no effect on cell death. In contrast, mTOR deletion, which attenuated UVB-induced phosphorylation of both S6K and the mTORC2 target AKTSer473, significantly increased apoptosis both in vivo and in keratinocyte cultures, in addition to reducing hyperproliferation following UVB irradiation. The role of mTORC2 in UVB-induced prosurvival signaling was verified in Rictor−/− mouse embryo fibroblasts, which lack functional mTORC2 and were more sensitive to UVB-induced apoptosis than controls. These studies show that mTORC1 and mTORC2 play unique but complementary roles in controlling proliferation and apoptosis in the skin. Our findings underscore the importance of both mTOR complexes in mediating UVB-induced signaling in keratinocytes and provide new insight into the pathogenesis of skin cancer. Cancer Prev Res; 5(12); 1394–404. ©2012 AACR.

Neoplastic growth is associated with increased polyamine biosynthetic activity and content. Tumor promoter treatment induces the rate-limiting enzymes in polyamine biosynthesis, ornithine decarboxylase (ODC), and S-adenosylmethionine decarboxylase (AdoMetDC), and targeted ODC overexpression is sufficient for tumor promotion in initiated mouse skin. We generated a mouse model with doxycycline (Dox)-regulated AdoMetDC expression to determine the impact of this second rate-limiting enzyme on epithelial carcinogenesis. TetO–AdoMetDC (TAMD) transgenic founders were crossed with transgenic mice (K5-tTA) that express the tetracycline-regulated transcriptional activator within basal keratinocytes of the skin. Transgene expression in TAMD/K5-tTA mice was restricted to keratin 5 (K5) target tissues and silenced upon Dox treatment. AdoMetDC activity and its product, decarboxylated AdoMet, both increased approximately 8-fold in the skin. This enabled a redistribution of the polyamines that led to reduced putrescine, increased spermine, and an elevated spermine:spermidine ratio. Given the positive association between polyamine biosynthetic capacity and neoplastic growth, it was somewhat surprising to find that TAMD/K5-tTA mice developed significantly fewer tumors than controls in response to 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate chemical carcinogenesis. Importantly, tumor counts in TAMD/K5-tTA mice rebounded to nearly equal the levels in the control group upon Dox-mediated transgene silencing at a late stage of tumor promotion, which indicates that latent viable initiated cells remain in AdoMetDC-expressing skin. These results underscore the complexity of polyamine modulation of tumor development and emphasize the critical role of putrescine in tumor promotion. AdoMetDC-expressing mice will enable more refined spatial and temporal manipulation of polyamine biosynthesis during tumorigenesis and in other models of human disease.

Upon Ras activation, ODC (ornithine decarboxylase) is markedly induced, and numerous studies suggest that ODC expression is controlled by Ras effector pathways. ODC is therefore a potential target in the treatment and prevention of Ras-driven tumours. In the present study we compared ODC mRNA translation profiles and stability in normal and Ras12V-transformed RIE-1 (rat intestinal epithelial) cells. While translation initiation of ODC increased modestly in Ras12V cells, ODC mRNA was stabilized 8-fold. Treatment with the specific mTORC1 [mTOR (mammalian target of rapamycin) complex 1] inhibitor rapamycin or siRNA (small interfering RNA) knockdown of mTOR destabilized the ODC mRNA, but rapamycin had only a minor effect on ODC translation initiation. Inhibition of mTORC1 also reduced the association of the mRNA-binding protein HuR with the ODC transcript. We have shown previously that HuR binding to the ODC 3′UTR (untranslated region) results in significant stabilization of the ODC mRNA, which contains several AU-rich regions within its 3′UTR that may act as regulatory sequences. Analysis of ODC 3′UTR deletion constructs suggests that cis-acting elements between base 1969 and base 2141 of the ODC mRNA act to stabilize the ODC transcript. These experiments thus define a novel mechanism of ODC synthesis control. Regulation of ODC mRNA decay could be an important means of limiting polyamine accumulation and subsequent tumour development.

Ornithine decarboxylase (ODC), the first enzyme of polyamine metabolism, is rapidly upregulated in response to agents that induce a pathological cardiac hypertrophy. Transgenic mice overexpressing ODC in the heart (MHC-ODC mice) experience a much more dramatic left ventricular hypertrophy in response to β-adrenergic stimulation with isoproterenol (ISO) compared to wild-type (WT) controls. ISO also induced arginase activity in transgenic hearts but not in controls. The current work studies the cooperation between the cardiac polyamines and L-arginine (L-Arg) availability in MHC-ODC mice. Although ISO-induced hypertrophy is well-compensated, MHC-ODC mice administered L-Arg along with ISO showed a rapid onset of systolic dysfunction and died within 48 h. Myocytes isolated from MHC-ODC mice administered L-Arg/ISO exhibited reduced contractility and altered calcium transients, suggesting an alteration in [Ca(2+)] homeostasis, and abbreviated action potential duration, which may contribute to arrhythmogenesis. The already elevated levels of spermidine and spermine were not further altered in MHC-ODC hearts by L-Arg/ISO treatment, suggesting alternative L-Arg utilization pathways lead to dysregulation of intracellular calcium. MHC-ODC mice administered an arginase inhibitor (Nor-NOHA) along with ISO died almost as rapidly as L-Arg/ISO-treated mice, while the iNOS inhibitor S-methyl-isothiourea (SMT) was strongly protective against L-Arg/ISO. These results point to the induction of arginase as a protective response to β-adrenergic stimulation in the setting of high polyamines. Further, NO generated by exogenously supplied L-Arg may contribute to the lethal consequences of L-Arg/ISO treatment. Since considerable variations in human cardiac polyamine and L-Arg content are likely, it is possible that alterations in these factors may influence myocyte contractility.

Ornithine decarboxylase (ODC) is the first and usually rate-limiting enzyme in the polyamine biosynthetic pathway. Under normal physiological conditions, polyamine content and ODC enzyme activity are highly regulated. However, the induction of ODC activity is an early step in neoplastic transformation. The studies described here use normal mouse keratinocytes (C5N cells), and spindle carcinoma cells (A5 cells) to explore the regulation of ODC in nonmelanoma skin cancer development. Previous results have shown that induction of ODC activity is both necessary and sufficient for the promotion of skin tumors. We see a marked increase in ODC enzyme activity in A5 cells compared with C5N keratinocytes, which correlates with a 4-fold stabilization of ODC mRNA. These data suggest that ODC is post-transcriptionally regulated in skin tumor development. Thus, we sought to investigate whether the ODC transcript interacts with the RNA-binding protein HuR, which is known to bind to and stabilize its target mRNAs. We show that HuR is able to bind to the ODC 3′-UTR in A5 cells but not in C5N cells. Immunofluorescence results reveal that HuR is present in both the nucleus and cytoplasm of A5 cells, whereas C5N cells exhibit strictly nuclear localization of HuR. Knockdown experiments in A5 cells showed that when HuR is depleted, ODC RNA becomes less stable, and ODC enzyme activity decreases. Together, these data support the hypothesis that HuR plays a causative role in ODC up-regulation during nonmelanoma skin cancer development by binding to and stabilizing the ODC transcript.

The present study was designed to provide a better understanding of the role played by AdoMetDC (S-adenosylmethionine decarboxylase), the key rate-controlling enzyme in the synthesis of spermidine and spermine, in controlling polyamine levels and the importance of polyamines in cardiac physiology. The αMHC (α-myosin heavy chain) promoter was used to generate transgenic mice with cardiac-specific expression of AdoMetDC. A founder line (αMHC/AdoMetDC) was established with a >100-fold increase in AdoMetDC activity in the heart. Transgene expression was maximal by 1 week of age and remained constant into adulthood. However, the changes in polyamine levels were most pronounced during the first week of age, with a 2-fold decrease in putrescine and spermidine and a 2-fold increase in spermine. At later times, spermine returned to near control levels, whereas putrescine and spermidine levels remained lower, suggesting that compensatory mechanisms exist to limit spermine accumulation. The αMHC/AdoMetDC mice did not display an overt cardiac phenotype, but there was an increased cardiac hypertrophy after β-adrenergic stimulation with isoprenaline (‘isoproterenol’), as well as a small increase in spermine content. Crosses of the αMHC/AdoMetDC with αMHC/ornithine decarboxylase mice that have a >1000-fold increase in cardiac ornithine decarboxylase were lethal in utero, presumably due to increase in spermine to toxic levels. These findings suggest that cardiac spermine levels are highly regulated to avoid polyamine-induced toxicity and that homoeostatic mechanisms can maintain non-toxic levels even when one enzyme of the biosynthetic pathway is greatly elevated but are unable to do so when two biosynthetic enzymes are increased.

A transgenic mouse line overexpressing a constitutively active mutant of MEK1, a downstream effector of Ras, driven by the keratin 14 (K14) promoter, has been used to test the hypothesis that ornithine decarboxylase (ODC) induction during tumor promotion following a single initiating event [i.e., the activation of the Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) pathway], is a necessary step in skin carcinogenesis. K14-MEK mice exhibit moderate hyperplasia, with spontaneous skin tumor development within 5 weeks of birth. Analysis of epidermis and dermis showed induction of MEK protein and ERK1/ERK2 phosphorylation, but no change in Akt-1, suggesting that the PI 3-kinase pathway, another pathway downstream of ras, is not activated. Examination of tumors revealed high levels of ODC protein and activity, indicating that activation of signaling cascades dependent on MEK activity is a sufficient stimulus for ODC induction. When K14-MEK mice were given α-difluoromethylornithine (DFMO), a suicide inactivator of ODC, in the drinking water from birth, there was a dramatic delay in the onset of tumor growth (∼6 weeks), and only 25% of DFMO-treated mice developed tumors by 15 weeks of age. All untreated K14-MEK mice developed tumors by 6 weeks of age. Treatment of tumor-bearing mice with DFMO reduced both tumor size and tumor number within several weeks. Tumor regression was the result of both inhibition of proliferation and increased apoptosis in tumors. The results establish ODC activation as an important component of the Raf/MEK/ERK pathway, and identify K14-MEK mice as a valuable model with which to study the regulation of ODC in ras carcinogenesis.

SUMMARY In a previous publication, we showed that a clinical trial of DL- � -difluoromethyl ornithine (DFMO), in combination with PCV (procarbazine, CCNU, vincristine) increased survival of patients with anaplastic gliomas (WHO III) but not glioblastoma multiforme (WHO IV). We believe that treatment outcome (survival) is inversely related to tumor ornithine decarboxylase (ODC) levels. To prove this, we needed to develop an assay to quantify ODC levels in formalin-fixed tumor tissues, which would enable a retrospective study of tumor biopsy specimens from the landmark clinical trial. We developed an assay using a specific polyclonal antibody coupled to an Alexa fluorescent dye. Transgenic MHCODC mice with differing levels of ODC in heart muscle were used to establish the relationship between mean gray-scale intensity and enzymatic ODC activity. We found a direct relationship between mean gray-scale intensity of the ODC antibody coupled to Alexa 647 dye and enzymatic activity. Preliminary analysis of a human glioma tissue array shows that tumor-specific variations in levels of ODC can be semiquantitated. We show that mean gray-scale intensity of astrocytoma:glioblastoma is 1:6 and of anaplastic astrocytoma:glioblastoma is 1:4. We also compared the intensity of antibody to Ki67 coupled with phycoerythrin simultaneously in cells but failed to see a relationship that crossed histologies. We conclude that we can measure levels of ODC in formalin-fixed tumor tissue using an antibody to ODC coupled to Alexa 647 dye, and this will enable us to conduct a future study to correlate survival of patients with gliomas of different histologies treated with DFMO to tumor ODC levels. (J Histochem Cytochem 52:1467–1474, 2004)

A transgenic mouse line expressing a truncated form of the ornithine decarboxylase (ODC) dominant-negative mutant K69A/C360A under the control of the keratin 6 promoter has been established (K6/ODCdn mice). These mice were backcrossed onto both the DBA/2J and C57BL/ 6J backgrounds for subsequent tumorigenesis experiments utilizing an initiation/promotion protocol. In short-term experiments, expression of the ODCdn protein product was induced in the epidermis within 24 h after application of the tumor promoter tetradecanoyl phorbol acetate (TPA) to the skin, and ODC activity in the epidermis of K6/ ODCdn mice was reduced by at least 75% compared with littermate controls. However, in tumorigenesis experiments utilizing a variety of initiator (7,12-dimethylbenz[a]anthracene; DMBA) and promoter (TPA) concentrations, K6/ ODCdn mice formed at least as many tumors as their littermate controls regardless of background strain. In experiments utilizing chrysarobin, a tumor promoter with a different mechanism of action than TPA, again there was no significant difference in tumor formation between K6/ODCdn mice and littermate controls. Similarly, when K6/ODCdn mice were crossed with K5/ODC mice, a transgenic line described previously which forms tumors without application of a promoting agent, double transgenic mice formed as many tumors as mice expressing the K5/ODC transgene alone. Analysis of epidermis following multiple TPA applications revealed a dramatic spike in ODC activity in both K6/ODCdn mice and non-transgenic mice after six applications, and western blot analysis suggested a stabilization of endogenous wild-type ODC in K6/ODCdn transgenic mice. ODC activity, endogenous protein and polyamines were also elevated in tumors from K6/ODCdn mice. The accumulation of endogenous ODC protein is most probably the result of competition from the transgenederived ODCdn protein for binding of antizyme, which is known to regulate ODC activity by stimulating degradation of the ODC protein.

Two lines of transgenic mice were produced with constitutive expression of antizyme-1 in the heart, driven from the cardiac α-myosin heavy chain promoter. The use of engineered antizyme cDNA in which nucleotide 205 had been deleted eliminated the need for polyamine-mediated frameshifting, normally necessary for translation of antizyme mRNA, and thus ensured the constitutive expression of antizyme. Antizyme-1 is thought to be a major factor in regulating cellular polyamine content, acting both to inhibit ornithine decarboxylase (ODC) activity and to target it for degradation, as well as preventing polyamine uptake. The two transgenic lines had substantial, but different, levels of antizyme in the heart, as detected by Western blotting and by the ability of heart extracts to inhibit exogenous purified ODC. Despite the high levels of antizyme, endogenous ODC activity was not completely abolished, with 10– 39% remaining, depending on the transgenic line. Additionally, a relatively small decrease (30–32%) in cardiac spermidine content was observed, with levels of putrescine and spermine unaffected. Interestingly, although the two lines of transgenic mice had different antizyme expression levels, they had almost identical cardiac polyamine content. When treated with a single acute dose of isoprenaline (isoproterenol), cardiac ODC activity and putrescine content were substantially increased (by 14-fold and 4.7-fold respectively) in non-transgenic littermate mice, but these increases were completely prevented in the transgenic mice from both founder lines. Prolonged exposure to isoprenaline also caused increases in cardiac ODC activity and polyamine content, as well as an increase in cardiac growth, in non-transgenic mice. Although the increases in cardiac ODC activity and polyamine content were prevented in the transgenic mice from both founder lines, the increase in cardiac growth was unaffected. These transgenic mice thus provide a valuable model system in which to study the importance of polyamine levels in cardiac growth and electrophysiology in response to stress.

It has long been known that polyamines play an essential role in the proliferation of mammalian cells, and the polyamine biosynthetic pathway may provide an important target for the development of agents that inhibit carcinogenesis and tumor growth. The rate-limiting enzymes of the polyamine pathway, ornithine decarboxylase (ODC) and S -adenosylmethionine decarboxylase (AdoMetDC), are highly regulated in the cell, and much of this regulation occurs at the level of translation. Although the 5′ leader sequences of ODC and AdoMetDC are both highly structured and contain small internal open reading frames (ORFs), the regulation of their translation appears to be quite different. The translational regulation of ODC is more dependent on secondary structure, and therefore responds to the intracellular availability of active eIF-4E, the cap-binding subunit of the eIF-4F complex, which mediates translation initiation. Cell-specific translation of AdoMetDC appears to be regulated exclusively through the internal ORF, which causes ribosome stalling that is independent of eIF-4E levels and decreases the efficiency with which the downstream ORF encoding AdoMetDC protein is translated. The translation of both ODC and AdoMetDC is negatively regulated by intracellular changes in the polyamines spermidine and spermine. Thus, when polyamine levels are low, the synthesis of both ODC and AdoMetDC is increased, and an increase in polyamine content causes a corresponding decrease in protein synthesis. However, an increase in active eIF-4E may allow for the synthesis of ODC even in the presence of polyamine levels that repress ODC translation in cells with lower levels of the initiation factor. In contrast, the amino acid sequence that is encoded by the upstream ORF is critical for polyamine regulation of AdoMetDC synthesis, and polyamines may affect synthesis by interaction with the putative peptide, MAGDIS.

Nucleus accumbens-1 (NAC1), a nuclear factor belonging to the BTB/POZ gene family, has emerging roles in cancer. We report here that NAC1 acts as a negative regulator of cellular senescence in transformed and nontransformed cells, and dysfunction of NAC1 induces senescence and inhibits its oncogenic potential. We show that NAC1 deficiency markedly activates senescence and inhibits proliferation in tumor cells treated with sublethal doses of γ-irradiation. In mouse embryonic fibroblasts from NAC1 knockout mice, following infection with a Ras virus, NAC1−/− cells undergo significantly more senescence and are either nontransformed or less transformed in vitro and less tumorigenic in vivo when compared with NAC1+/+ cells. Furthermore, we show that the NAC1-caused senescence blunting is mediated by ΔNp63, which exerts its effect on senescence through p21, and that NAC1 activates transcription of ΔNp63 under stressful conditions. Our results not only reveal a previously unrecognized function of NAC1, the molecular pathway involved and its impact on pathogenesis of tumor initiation and development, but also identify a novel senescence regulator that may be exploited as a potential target for cancer prevention and treatment. Cancer Res; 72(16); 4262–75. ©2012 AACR.

Mammalian S-adenosylmethionine decarboxylase (AdoMetDC) is known to be regulated by putrescine in two ways: (a) acceleration of the rate of conversion of the proenzyme into the mature enzyme in a reaction that forms the pyruvate prosthetic group and (b) activation of the mature enzyme activity. To determine sites of putrescine interaction with AdoMetDC, putrescine stimulation of both proenzyme processing and catalytic activity was tested with mutant AdoMetDCs in which specific amino acid residues, conserved between mammalian and yeast AdoMetDCs, had been altered by site-directed mutagenesis. Mutations E178Q or E256Q (and the previously reported mutation E11Q (Stanley, B. A., and Pegg, A. E. (1991) J. Biol. Chem. 266, 18502-18506)) abolished stimulation by putrescine without an effect on the processing rate in the absence of putrescine. Mutations E11K, as well as Y112A and L259Stop, completely abolished processing regardless of putrescine concentration, whereas mutation E133Q conferred an absolute putrescine requirement for processing to occur. Mutation E132Q, E135Q, E183Q, or D185N had no effect on proenzyme processing. The effects of mutations on enzyme activity were determined using AdoMetDC protein produced in Escherichia coli and purified by affinity chromatography. Mutation E11Q completely inactivated the enzyme, mutation E133Q reduced the catalytic constant by > 10(4), and mutation E256Q produced a 20-fold decrease. Putrescine did not stimulate the activity of mutants E178Q and E256Q but did activate mutants E133Q and E183Q. It is concluded that residues Glu-11, Glu-178, and Glu-256 are critical residues in the putrescine stimulation of AdoMetDC proenzyme processing and that Glu-178 and Glu-256 are critical for putrescine stimulation of AdoMetDC catalytic activity.

Myoblast differentiation into multinuclear myotubes implies the slow-down of their proliferative drive and the expression of myogenin, an early marker of myogenic differentiation. Natural polyamines-such as putrescine, spermidine and spermine-are low molecular weight organic polycations, well known as mediators involved in cell homeostasis. Many evidences in the literature point to their role in driving cellular differentiation processes. Here, we studied how polyamines may affect the differentiation of the myogenic cell line H9c2 into the muscle phenotype. Cell cultures were committed via a 7-day treatment with insulin which induced increase in the activity of ornithine decarboxylase, the first enzyme in the polyamine biosynthetic pathway, consistent with myogenic differentiation. To evaluate the role of polyamines in the differentiation process, cells were transfected with a plasmid overexpressing a stable ornithine decarboxylase, under control of a constitutive promoter. Overexpressing cells spontaneously differentiate into myotubes, without the need for induction with insulin; multinuclear myotubes and myogenin expression were apparent within 2 days of confluency of cultures. Polyamine depletion-by means of alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase-abolished the differentiation process. These observations support the evidence that polyamines are a key step involved in differentiation of muscle cells.

The role of ornithine decarboxylase (ODC) in polyamine metabolism has long been established, but the exact source of ornithine has always been unclear. The arginase enzymes are capable of producing ornithine for the production of polyamines and may hold important regulatory functions in the maintenance of this pathway. Utilizing our unique set of arginase single and double knockout mice, we analyzed polyamine levels in the livers, brains, kidneys, and small intestines of the mice at 2 wk of age, the latest timepoint at which all of them are still alive, to determine whether tissue polyamine levels were altered in response to a disruption of arginase I (AI) and II (AII) enzymatic activity. Whereas putrescine was minimally increased in the liver and kidneys from the AII knockout mice, spermidine and spermine were maintained. ODC activity was not greatly altered in the knockout animals and did not correlate with the fluctuations in putrescine. mRNA levels of ornithine aminotransferase (OAT), antizyme 1 (AZ1), and spermidine/spermine- N1-acetyltransferase (SSAT) were also measured and only minor alterations were seen, most notably an increase in OAT expression seen in the liver of AI knockout and double knockout mice. It appears that putrescine catabolism may be affected in the liver when AI is disrupted and ornithine levels are highly reduced. These results suggest that endogenous arginase-derived ornithine may not directly contribute to polyamine homeostasis in mice. Alternate sources such as diet may provide sufficient polyamines for maintenance in mammalian tissues.

The purpose of this study was to assess the relationship between progression-free survival (PFS) in patients treated with DFMO + PCV (procarbazine, CCNU, vincristine) chemotherapy for malignant gliomas with tumor cell ornithine decarboxylase (ODC) activity. Formalin-fixed slides were obtained for study patients with anaplastic gliomas (AGs) and glioblastoma treated on protocol DM92-035. ODC levels were measured using an antibody to ODC coupled to Alexa 647 dye (Ab-ODC-Alexa 647). Ab-ODC-Alexa 647 intensity in transgenic murine hearts of differing ODC activity was used to calculate ODC activity in tumor cell nucleoplasm. In total, tumor specimens for 31 of 114 (27%) patients treated on the AG strata and 10 patients from the GBM strata were obtained. We found that tumor ODC level heterogeneity increased with increasing tumor malignancy. In a Cox proportional hazards model, PFS was found to be inversely related to median tumor ODC activity, with an unadjusted hazard ratio for median ODC group (3.3 vs./=3.3 nmol/30 min/mug protein) of 5.8 (p0.0001); a median PFS of 522 weeks for patients with AGs with median ODC activity/= 3.3 and 31 weeks for the 8 AG and 10 glioblastoma patients with ODC activity3.3 nmol/30 min/mug protein. Of AG tumors in which ODC activity was evaluated, 26% had ODC levels3.3 nmol/30 min/mug protein. This study shows that Ab-ODC-Alexa 647 fluorescence intensity can be used as a surrogate marker of ODC biochemical activity in AGs and can predict PFS to DFMO-based chemotherapy.

Ornithine decarboxylase (ODC) is the first and generally rate-limiting enzyme in polyamine biosynthesis. Deregulation of ODC is critical for oncogenic growth, and ODC is a target of Ras. These experiments examine translational regulation of ODC in RIE-1 cells, comparing untransformed cells with those transformed by an activated Ras12V mutant. Analysis of the ODC 5′ untranslated region (5′UTR) revealed four splice variants with the presence or absence of two intronic sequences. All four 5′UTR species were found in both cell lines; however, variants containing intronic sequences were more abundant in Ras-transformed cells. All splice variants support internal ribosome entry site (IRES)–mediated translation, and IRES activity is markedly elevated in cells transformed by Ras. Inhibition of Ras effector targets indicated that the ODC IRES element is regulated by the phosphorylation status of the translation factor eIF4E. Dephosphorylation of eIF4E by inhibition of mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) or the eIF4E kinase Mnk1/2 increases ODC IRES activity in both cell lines. When both the Raf/MEK/ERK and phosphatidylinositol 3-kinase/mammalian target of rapamycin pathways are inhibited in normal cells, ODC IRES activity is very low and cells arrest in G1. When these pathways are inhibited in Ras-transformed cells, cell cycle arrest does not occur and ODC IRES activity increases, helping to maintain high ODC activity. [Cancer Res 2007;67(10):4834–42]

Elevated polyamine content and increased ornithine decarboxylase (ODC) activity have been associated with neoplastic growth in numerous animal models and human tissues. Antizyme (AZ) is a negative regulator of polyamine metabolism that inhibits ODC activity, stimulates ODC degradation, and suppresses polyamine uptake. Preliminary evidence, obtained from transgenic mice with tissue specific overexpression of AZ indicates that tumor development can be suppressed by AZ. To extend these studies, we have examined the effect of keratin 5 (K5)- or K6-driven AZ transgenes on 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) chemical carcinogenesis of the skin, in promotion-resistant C57BL/6 and promotion-sensitive DBA/2 mice. On both genetic backgrounds, K6-AZ mice showed a reduction in tumor multiplicity, with 85% fewer tumors than wild-type controls on the C57BL/6 background and 50% fewer tumors on the DBA/2 background. K5-AZ mice developed 50% fewer tumors than controls on both backgrounds. The percent of mice with tumors and tumor size were also reduced in the K5-AZ and K6-AZ groups. Tumor and TPA-treated skin sections from K6-AZ mice exhibited the strongest AZ expression, with localization mainly in suprabasal keratinocytes. K6-AZ mice also had slightly reduced cell proliferation rates in tumors and TPA-treated skin. The lack of a more pronounced effect on cell proliferation is probably explained by the observation that AZ staining did not colocalize with proliferating cell nuclear antigen (PCNA), a marker for the proliferative compartment. These studies demonstrate a tumor-suppressive effect of AZ in C57BL/6 and DBA/2 mice, and confirm the importance of ODC and polyamines in tumor development.

The greatest environmental risk factor for developing non-melanoma skin cancer (NMSC) is ultraviolet B (UVB) radiation. Both complexes of the mammalian target of rapamycin (mTOR) signaling pathway, the rapamycin-sensitive mTOR complex 1 (mTORC1) and the rapamycin-resistant mTOR complex 2 (mTORC2), are stimulated in response to UVB. Cell survival in UVB-induced keratinocyte carcinogenesis has been shown to be mTORC2-depedent. The tumor suppressor FOXO3a, a member of the Forkhead box family of proteins, is an important transcription factor involved in regulating cell survival. FOXO3a activity is attenuated as a result of AKT activation, which is downstream of mTORC2. UVB has been shown to activate AKT through the mTORC2 pathway; however, the role of FOXO3a in UVB-induced apoptosis in keratinocytes has yet to be studied. To investigate the role of mTORC2 and FOXO3a in UVB-induced apoptosis, we utilized both human keratinocytes (HaCaT cells) as well as inducible Rictor-deficient mouse embryonic fibroblasts (iRictKO cells) following treatment with 4-hydroxy-tamoxifen (4OHT). We also employed the use of inhibitors and shRNA targeting Rictor or mSIN1. The Rictor and mSIN1 proteins are key structural components unique to mTORC2 and their removal dramatically lowers mTORC2 activity. Finally, we compared the effects of shRNA targeting FOXO3a with the expression of a constitutively active mutant FOXO3a. For all the experiments described, cells were exposed to an apoptotic dose of UVB and allowed to incubate for defined experimentally established intervals. Our results show that there is an mTORC2-dependent regulation of FOXO3a expression, localization, and activity upon UVB-irradiation. Through western analysis we demonstrate that UVB exposure promotes the phosphorylation of FOXO3a by AKT in cells with intact mTORC2 signaling, which is repressed when mTORC2 is inhibited. This phosphorylation has been shown in other systems to reduce FOXO3a activity by causing a cytoplasmic localization of the protein. In agreement with this, we observed a rapid cytoplasmic localization of FOXO3a and decreased FOXO3a promoter activation in response to UVB, which is rescued following disruption of mTORC2 signaling. Additionally, we find that FOXO3a protein expression is reduced upon UVB-irradiation when mTORC2-signaling is reduced. These data support previous findings that suggest the mTORC2-driven FOXO3a cytoplasmic sequestration protects FOXO3a from dephosphorylation and degradation. Taken together, these studies provide strong evidence that mTORC2 plays a critical role in regulating FOXO3a expression, localization and activity following UVB-irradiation. In addition, our results support the hypothesis that the mTORC2-dependent UVB-induced apoptosis is due at least in part to FOXO3a. Supported by ES19242 (LMS) Citation Format: Robert P. Feehan, Lisa M. Shantz. The role of the mTORC2-dependent regulation of FOXO3a in UVB-induced apoptosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 27. doi:10.1158/1538-7445.AM2015-27

The activity of ornithine decarboxylase (ODC(1)), the first enzyme in polyamine biosynthesis, is induced during carcinogenesis by a variety of oncogenic stimuli. Intracellular levels of ODC and the polyamines are tightly controlled during normal cell growth, and regulation occurs at the levels of transcription, translation and protein degradation. Several known proto-oncogenic pathways appear to control ODC transcription and translation, and dysregulation of pathways downstream of ras and myc result in the constitutive elevation of ODC activity that occurs with oncogenesis. Inhibition of ODC activity reverts the transformation of cells in vitro and reduces tumor growth in several animal models, suggesting high levels of ODC are necessary for the maintenance of the transformed phenotype. The ODC irreversible inactivator DFMO has proven to be not only a valuable tool in the study of ODC in cancer, but also shows promise as a chemopreventive and chemotherapeutic agent in certain types of malignancies.

The antizyme inhibitor was discovered as a protein that binds to the regulatory protein antizyme and inhibits the ability of antizyme to interact with the enzyme ornithine decarboxylase (ODC). Blocking antizyme activity subsequently leads to increased intracellular levels of ODC and increased ODC enzymatic activity. We now report that antizyme inhibitor is a positive modulator of cell growth. Overexpression of antizyme inhibitor in NIH-3T3 mouse fibroblasts or in AT2.1 Dunning rat prostate carcinoma cells resulted in an increased rate of cell proliferation and an increase in saturation density of the cultured cells. This was accompanied by an increase in intracellular levels of the polyamine putrescine. In AT2.1 cells, antizyme inhibitor overexpression also increased the ability of the cells to form foci when grown under anchorage-independent conditions. In order to determine the role of antizyme on antizyme inhibitor activity we created an antizyme inhibitor mutant, AZIΔ117-140, which lacks the putative antizyme-binding domain. We show that this mutant fails to bind to antizyme, but remains capable of inducing increased rates of cell proliferation, suggesting that antizyme inhibitor has antizyme-independent functions. Silencing antizyme inhibitor expression leads to diminished levels of cyclin D1 and to reduced cell proliferation. Antizyme inhibitor is capable of preventing cyclin D1 degradation, and this effect is at least partially independent of antizyme. We show that wild-type antizyme inhibitor and the AZIΔY mutant are capable of direct interaction with cyclin D1 suggesting a potential mechanism for the antizyme-independent effects. Together, our data suggest a novel function for antizyme inhibitor in cellular growth control.

To test the hypothesis that suppression of ornithine decarboxylase (ODC) activity blocks the promotion of target cells in the outer root sheath of the hair follicle initiated by Raf/MEK/ERK activation, we crossed mice overexpressing an activated MEK mutant in the skin (K14-MEK mice) with two transgenic lines overexpressing antizyme (AZ), which binds to ODC and targets it for degradation. K14-MEK mice develop spontaneous skin tumors without initiation or promotion. These mice on the ICR background were crossed with K5-AZ and K6-AZ mice on both the carcinogenesis-resistant C57BL/6 background and the sensitive DBA/2 background. Expression of AZ driven by either the K5 or K6 promoter along with K14-MEK dramatically delayed tumor incidence and reduced tumor multiplicity on both backgrounds compared with littermates expressing the MEK transgene alone. The effect was most remarkable in the MEK/K6-AZ mice from the ICR/D2 Fl cross, where double transgenic mice averaged less than one tumor per mouse for more than 8 weeks, while K14-MEK mice averaged over 13 tumors per mouse at this age. Putrescine was decreased in MEK/AZ tumors, while spermidine and spermine levels were unaffected, suggesting that the primary role played by AZ in this system is to inhibit putrescine accumulation. MEK/AZ tumors did not show evidence of apoptosis, but there was a 15-20% decrease in S-phase cells and a 40-60% decrease in mitotic cells in MEK/AZ tumors. These results indicate that the principal effect of AZ may be to slow cell growth primarily by increasing G 2 /M transit time.

none 12 Apoptotic cell death of cardiomyocytes is involved in several cardiovascular diseases including ischemia, hypertrophy, and heart failure. The polyamines putrescine, spermidine, and spermine are polycations absolutely required for cell growth and division. However, increasing evidence indicates that polyamines, cell growth and cell death can be tightly connected. In this paper we have studied the involvement of polyamines in apoptosis of H9c2 cardiomyoblasts in a model of simulated ischemia. H9c2 cells were exposed to a condition of simulated ischemia, consisting of hypoxia plus serum deprivation, that induces apoptosis. The activity of ornithine decarboxylase, the rate limiting enzyme of polyamine biosynthesis that synthesizes putrescine, is rapidly and transiently induced in ischemic cells, reaching a maximum after 3 h, and leading to increased polyamine levels. Pharmacological inhibition of ornithine decarboxylase by -difluoromethylornithine (DFMO) depletes H9c2 cardiomyoblasts of polyamines and protects the cells against ischemia-induced apoptosis. DFMO inhibits several of the molecular events of apoptosis that follow simulated ischemia, such as the release of cytochrome c from mitochondria, caspase activation, downregulation of Bcl-xl, and DNA fragmentation. The protective effect of DFMO is lost when exogenous putrescine is provided to the cells, indicating a specific role of polyamine synthesis in the development of apoptosis in this model of simulated ischemia. In cardiomyocytes obtained from transgenic mice overexpressing ornithine decarboxylase in the heart, caspase activation is dramatically increased following induction of apoptosis, with respect to cadiomyocytes from control mice, confirming a proapoptotic effect of polyamines. It is presented for the first time evidence of the involvement of polyamines in apoptosis of ischemic cardiac cells and the beneficial effect of DFMO treatment. In conclusion this finding may suggest novel pharmacological approaches for the protection of cardiomyocytes injury caused by ischemia. Tantini B.; Fiumana E.; Cetrullo S.; Pignatti C.; Bonavita F.; Shantz LM.; Giordano E.; Muscari C; Flamigni F.; Guarnieri C.; Stefanelli C.; Caldarera CM. Tantini B.; Fiumana E.; Cetrullo S.; Pignatti C.; Bonavita F.; Shantz LM.; Giordano E.; Muscari C; Flamigni F.; Guarnieri C.; Stefanelli C.; Caldarera CM.

Cardiac muscle hypertrophy is one of the most important compensatory responses of the heart to multiple stresses that may be placed on it. If the stress is not relieved, sustained hypertrophy may progress to dysfunction and heart failure. The polyamines putrescine, spermidine, and spermine increase within hours of various types of experimentally induced cardiac hypertrophy, along with ornithine decarboxylase (ODC) gene expression. Several animal models have implicated ODC induction as an important factor in the development of hypertrophy, particularly in response to β-adrenergic stimulation. Novel transgenic mouse lines that overexpress several enzymes of polyamine metabolism in the heart have been generated in recent years, and crosses of these lines have pointed to decarboxylated adenosylmethionine and its control by S-adenosylmethionine decarboxylase as another important element in maintaining cardiac polyamine homeostasis. The activity of arginase is thought to play a regulatory role in the biosynthesis of both nitric oxide (NO) and polyamines, and NO deficiency has been linked to the development of cardiac hypertrophy. Genetically altered mouse lines with changes in arginine and NO metabolism in the heart are available, many of which possess cardiac abnormalities. These models will provide a valuable means to address the interdependence of arginine and ornithine metabolism in the development of myocardial hypertrophy and failure. Use of these tools may lead to a better understanding of the control of the signaling pathways that include the polyamines, arginine, and NO, allowing future work to focus on the interactions between these pathways in the development of heart disease.

A widespread increase in SpmS (spermine synthase) activity has been produced in transgenic mice using a construct in which the human SpmS cDNA was placed under the control of a composite CMV-IE (cytomegalovirus immediate early gene) enhancer–chicken β-actin promoter. Four separate founder CAG/SpmS mice were studied. Transgenic expression of SpmS was found in all of the tissues examined, but the relative SpmS activities varied widely according to the founder animal and the tissue studied. Very large increases in SpmS activity were seen in many tissues. SpdS (spermidine synthase) activity was not affected. Although there was a statistically significant decline in spermidine content and increase in spermine, the alterations were small compared with the increase in SpmS activity. These results provide strong support for the concept that the levels of the higher polyamines spermidine and spermine are not determined only by the relative activities of the two aminopropyltransferases. Other factors such as availability of the aminopropyl donor substrate decarboxylated S-adenosylmethionine and possibly degradation or excretion must also influence the spermidine/spermine ratio. No deleterious effects of SpmS overexpression were seen. The mice had normal growth, fertility and behaviour up to the age of 12 months. However, breeding the CAG/SpmS mice with MHC (α-myosin heavy chain)/AdoMetDC (S-adenosylmethionine decarboxylase) mice, which have a large increase in S-adenosylmethionine decarboxylase expression in heart, was lethal. In contrast, breeding the CAG/SpmS mice with MHC/ODC (L-ornithine decarboxylase) mice, which have a large increase in cardiac ornithine decarboxylase expression, had a protective effect in preventing the small decrease in viability of the MHC/ODC mice.

Transgenic mice expressing proteins altering polyamine levels in a tissue-specific manner have considerable promise for evaluation of the roles of polyamines in normal, hypertrophic and neoplastic growth. This short review summarizes the available transgenic models. Mice with large increases in ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase or antizyme, a protein regulating polyamine synthesis by reducing polyamine transport and ODC in the heart, have been produced using constructs in which the protein is expressed from the alpha -myosin heavy-chain promoter. These mice are useful in studies of the role of polyamines in hypertrophic growth. Expression from keratin promoters has been used to target increased synthesis of ODC, spermidine/spermine-N(1)-acetyltransferase (SSAT) and antizyme in the skin. Such expression of ODC leads to an increased sensitivity to chemical and UV carcinogenesis. Expression of antizyme inhibits carcinogenesis in skin and forestomach. Expression of SSAT increases the incidence of skin papillomas and their progression to carcinomas in response to a two-stage carcinogenesis protocol. These results establish the importance of polyamines in carcinogenesis and neoplastic growth and these transgenic mice will be valuable experimental tools to evaluate the importance of polyamines in mediating responses to oncogenes and studies of cancer chemoprevention.

Both ornithine decarboxylase (ODC) and nitric oxide synthase (NOS) activities rely on the availability of the common substrate L-arginine, which is directly processed by NOS to nitric oxide (NO) and L-citrulline. Alternatively, arginine is acted on by arginase in the urea cycle to produce ornithine, which then enters polyamine biosynthesis. Evidence in the literature points out that NO is able to inhibit ODC and polyamines can inhibit NOS. Both of these observations seem to indicate that the two metabolic pathways may crosstalk in regulating complex organic functions and the heart is a possible target organ for this interplay. It has been demonstrated that upregulation of ODC activity is critical in the hypertrophic myocardial response, which is also consistent with observations involving conditions of nitric oxide deficiency.