Elevated levels of ornithine decarboxylase cooperate with Raf/ERK activation to convert normal keratinocytes into invasive malignant cells.

Ornithine decarboxylase (ODC) overexpression coupled with activated Ras is fully sufficient to oncogenically transform primary keratinocytes. To determine the Ras effector pathways that represent the minimal essential contribution to full oncogenic transformation in this context, we evaluated the cooperativity of different Ras effector mutants with overexpressed ODC in an in vivo tracheal xenotransplantation assay for epithelial cell invasiveness. Primary keratinocytes, isolated from either K6/ODC transgenic mouse skin (expressing increased ODC) or from normal littermate skin were infected with retrovirus producing an activated RasV12 or partial loss-of-function effector mutants of RasV12 that selectively induce only the Raf/ERK, RalGDS, or the PI3-kinase signaling pathway. Whereas keratinocytes expressing a fully activated RasV12 are not invasive in tracheal xenotransplants, ODC-overexpressing keratinocytes acquire an invasive phenotype with additional expression of either RasV12 or activation of the Raf/ERK pathway. Independent of a mutated ras, elevated levels of ODC activate the Akt/mTOR signaling pathway as well as the Rho/Rac pathway in primary keratinocytes. Thus, Raf/ERK signaling is sufficient to cooperate with increased ODC activity in the conversion of normal keratinocytes to invasive cells. In order to promote invasiveness in keratinocytes, elevated levels of ODC may cooperate with Raf/ERK via activation of the Akt and Rho/Rac signaling pathway.

Polyamines regulate gap junction communication in connexin 43-expressing cells.

The control of cell-cell communication through gap junctions is thought to be crucial in normal tissue function and during various stages of tumorigenesis. However, few natural regulators of gap junctions have been found. We show here that increasing the activity of ornithine decarboxylase, or adding polyamines to the outside of cells, increases the level of gap junction communication between various epithelial cells. Conversely, reduction of ornithine decarboxylase activity decreases the level of gap junction communication. This regulation is dependent upon the expression of connexin 43 (Cx43 or Cxalpha1), which is a major connexin expressed in many different cell types, and involves an increase in Cx43 and its cellular re-distribution.

Inhibition of ornithine decarboxylase (ODC) decreases tumor vascularization and reverses spontaneous tumors in ODC/Ras transgenic mice.

We have shown that ornithine decarboxylase (ODC) overexpression in the skin of TG.AC v-Ha-ras transgenic mice induces the formation of spontaneous skin carcinomas. Treatment of ODC/Ras double transgenic mice with alpha-difluoromethylornithine (DFMO), a specific inhibitor of ODC enzyme activity, causes a rapid regression of these spontaneous tumors. DFMO treatment led to dramatic decreases in ODC activity and putrescine levels, but v-Ha-ras expression was not affected in the regressed tumors. Moreover, cyclin D1 continued to be strongly expressed in the basal epithelial cells of regressed tumors, and there was no decrease in the proliferative index of these same tumor cells. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling analyses revealed increased DNA fragmentation in DFMO regressed tumors compared with similarly sized spontaneous tumors from ODC/Ras transgenic mice not treated with DFMO. Moreover, the blood vessel count was significantly decreased in regressed tumors within the first four days of DFMO treatment. The decreased vasculature in DFMO regressed tumors was not attributable to altered expression of murine vascular endothelial growth factor (VEGF) isoforms. Elevated levels of ODC activity in the skin of K6/ODC transgenic mice increased the dermal vascularization compared with that in nontransgenic normal littermates. Our results suggest that ODC stimulates an angiogenic factor(s) other than VEGF and/or may play a key role in a cell survival effector pathway of Ras that is independent of a Ras-induced proliferation pathway.

K6/ODC transgenic mice as a sensitive model for carcinogen identification.

Ornithine decarboxylase (ODC), an important enzyme in the polyamine biosynthetic pathway, is aberrantly regulated in many epithelial tumors of rodents and humans. In murine skin, it has been shown that ODC overexpression provides a sufficient condition for tumor promotion. Therefore, we hypothesized that K6/ODC transgenic mice in which ODC overexpression was targeted to hair follicle keratinocytes might provide a sensitive model for identifying genotoxic carcinogens. Ten known carcinogens or noncarcinogens have been tested in the model so far and results are highly concordant with 2-year rodent bioassays (100% concordant). More importantly, each of two chemicals tested that is recognized as a human carcinogen was identified as a carcinogen in K6/ODC transgenic mice. In addition, 7, 12-dimethylbenz(a)anthracene (DMBA) dose response studies indicated that even at a very low dose, 2 nmol, a high percentage of mice (50%) had already developed tumors 8 weeks after treatment. We conclude that the K6/ODC transgenic mouse model is very sensitive to topical application of genotoxic carcinogens and could therefore be a useful mouse model for carcinogen identification and chemical risk assessment.

High levels of intracellular polyamines promote histone acetyltransferase activity resulting in chromatin hyperacetylation.

Polyamines stimulate expression of a variety of genes, including many implicated in cell proliferation. Indeed, aberrant expression of ornithine decarboxylase (ODC), a rate-limiting enzyme in polyamine biosynthesis, plays a causal role in tumorigenesis. Gene activity is influenced by dynamic changes in acetylation of nucleosomal histones. Although polyamines influence the histone acetyltransferase and deacetylase activities in cell-free systems, their ability to modulate these enzymes in live cells has never been established. To examine the effects of elevated intracellular levels of ODC and polyamines on gene transcription and histone acetylation, cells were infected with a retrovirus containing a cDNA for ODC. ODC overexpression potentiated the stimulatory effects of histone deacetylase inhibitors on reporter gene expression beyond that promoted by ODC or inhibitor treatment alone. Indeed, elevated intracellular levels of ODC promoted hyperacetylation of histones in several epidermal and fibroblast cell types. The ODC-mediated increase in acetylated histones was abrogated when cells were treated with alpha-difluoromethylornithine, a specific inhibitor of ODC activity, implying a distinct role for polyamines. Specifically, polyamines were found to enhance the action of histone acetyltransferases either directly or indirectly. Our studies document effects of elevated intracellular polyamine levels on histone acetylation in proliferating cells, suggesting a mechanism by which altered polyamine biosynthesis contributes to aberrant expression of genes, facilitating tumor growth. In addition, these studies may have implications for the development of drugs designed to regulate enzymes that modify the acetylation status of histones.

Effect of elevated levels of ornithine decarboxylase on cell cycle progression in skin.

By crossing TG.AC v-Ha-ras and K6/ODC transgenic mice, we found previously that an activated ras and follicular ornithine decarboxylase (ODC) overexpression cooperate to generate spontaneous tumors in the skin. Cellular proliferation was dramatically increased in the K6/ODC transgenic skin, as evidenced by elevated proliferating cell nuclear antigen and Ki67 expression compared with nontransgenic littermates. Keratinocytes isolated from transgenic skin also displayed increased clonal growth. Paradoxically, expression of the growth inhibition-associated proteins p53, p21Waf1, p27Klp1, and Bax was increased with ODC overexpression in the skin. ODC overexpression did not affect cyclin D/cyclin-dependent kinase 4 (Cdk4)-dependent phosphorylation of retinoblastoma protein but stimulated cyclin E/Cdk2 and cyclin A/Cdk2-associated kinase activity, with minimal effect on the levels of these proteins. Thus, ODC/polyamine-induced activation of cyclin E/Cdk2 and cyclin A/Cdk2-associated kinase activity may cooperate with the ras induction of cyclin D/Cdk4/6-associated retinoblastoma protein phosphorylation to not only stimulate proliferation but ultimately contribute to tumor development.

Co-operation between follicular ornithine decarboxylase and v-Ha-ras induces spontaneous papillomas and malignant conversion in transgenic skin.

Ornithine decarboxylase (ODC) is aberrantly regulated in tumor cells and results in high basal levels of ODC and polyamines in many epithelial tumors. To determine if elevated ODC/polyamine levels can co-operate with a mutant Ha-ras gene in mouse skin tumorigenesis, double transgenic mice were generated by breeding K6/ODC transgenic mice with TG.AC v-Ha-ras transgenic mice. A K6 keratin promoter drives the ODC transgene in K6/ ODC transgenic mice, which results in elevated ODC/ polyamine levels directed to the outer root sheath cells of hair follicles. TG.AC transgenic mice carry a v-Ha-ras transgene while still retaining two normal c-Ha-ras alleles. Transgenic mice that possess only the K6/ODC or the v-Ha-ras transgene did not develop tumors unless treated with either a carcinogen or a tumor promoter, respectively. However, a high percentage of double transgenic mice possessing both the K6/ODC and v-Ha-ras transgenes developed spontaneous tumors. All tumors were well-differentiated keratoacanthomas, some of which progressed to carcinomas within 2 months. The development and the maintenance of these ODC/ras tumors was ODC-dependent since alpha-difluoromethylornithine (DFMO), a specific ODC inhibitor, prevented the formation and caused the regression of these tumors. These findings indicate that ODC overexpression and an activated Ha-ras are sufficient to produce a high rate of malignant transformation in an animal model. The ODC/ras double transgenic mouse provides a simple in vivo model without the use of chemical carcinogens or tumor promoters in which to test downstream effectors that play a key role in mediating the development of epithelial tumors resulting from the cooperation between ODC and v-Ha-ras.

Ornithine decarboxylase overexpression leads to increased epithelial tumor invasiveness.

Ornithine decarboxylase (ODC) overexpression cooperates with genetic lesions such as an activated c-rasHa to enhance epithelial tumorigenesis. To assess the invasiveness of ODC-overexpressing cells, two noninvasive epidermal cell lines, nontumorigenic BK-1 cells, and the papilloma-derived cell line SP-1 were infected with a replication-defective retrovirus that overexpresses ODC, inoculated into deepithelialized rat tracheas, and transplanted into athymic nude mice. After 5 weeks, ODC-overexpressing BK-1 cells remained localized on the luminal surface of the tracheal xenotransplants, whereas the ODC-overexpressing SP-1 cells were extremely invasive, with the whole tracheal wall penetrated. This invasiveness of ODC-overexpressing SP-1 cells was accompanied by elevated proteinase expression, including increased urokinase plasminogen activator activity in ODC-overexpressing cells and elevated stromelysin-1 mRNA expression in the stromal cells of invaded tracheal transplants.

Ornithine decarboxylase expression leads to translocation and activation of protein kinase CK2 in vivo.

Ornithine decarboxylase (ODC) is the key initial enzyme in the biosynthesis of polyamines. Since polyamines have been shown to enhance protein kinase CK2 activity in vitro, ODC was overexpressed to examine the role of polyamines in CK2 regulation in vivo. Infection of Balb/MK cells with an ODC retrovirus to elevate ODC and polyamine levels increased overall protein phosphorylation as well as CK2 protein levels and enzyme activity in mimosine- or nocodazole- arrested cells. Immunofluorescence microscopy and enzyme analyses of subcellular fractions from ODC-overexpressing cells demonstrated translocation of CK2 from the cytoplasm to the nucleus with no apparent loss of cytoplasmic CK2 activity, suggesting polyamine activation of the remaining cytoplasmic enzyme. Similarly, K6/ODC transgenic mice exhibited higher ODC and CK2 enzyme activities than their normal littermates. ODC-immunostained cells in the transgenic skin also stained intensely for CK2 protein. Primary cultures of K6/ODC keratinocytes also exhibited increased ODC and CK2 enzyme activities compared with those from normal littermates. However, the addition of difluoromethylornithine, a specific ODC inhibitor, to the transgenic keratinocytes reduced both intracellular polyamine levels and CK2 enzyme activity. These results suggest that polyamines regulate the CK2 enzyme by affecting its cellular distribution as well as its enzyme activity and levels.

Elevated cellular polyamine levels enhance promoter activity in vivo.

Polyamines are small polycationic molecules that have been implicated in cell growth, differentiation and transformation. A possible mechanism for their involvement in these processes is via their influence on the expression of growth promoting genes. In this study we used an ornithine decarboxylase (ODC) overexpressing retroviral system to achieve high intracellular levels of polyamines in epidermal cells. We then looked at the effect this environment had on transcription from several promoter-reported gene constructs, including c-myc and ODC itself. In transient transfection studies elevated polyamines significantly enhanced the transcriptional activity of all promoters tested. In addition, reporter gene expression was induced approximately 3 fold in stable transformants containing an integrated c-myc or ODC promoter-reporter gene construct suggesting that elevated levels of polyamines, such as those found in transformed cells, have a stimulatory effect on gene expression.

Increased frequency of spontaneous skin tumors in transgenic mice which overexpress ornithine decarboxylase.

Ornithine decarboxylase, a critical regulatory enzyme for polyamine biosynthesis, is highly inducible by growth-promoting stimuli in mouse epidermis but the enzyme level is only transiently elevated due to rapid turnover of the protein. Here we report that constitutive overexpression of the enzyme in the skin of transgenic mice causes several phenotypic abnormalities. Effects observed include development of dermal follicular cysts, excessive skin wrinkling, enhanced nail growth, alopecia, and spontaneous tumor development. These results indicate that up-regulation of polyamine biosynthesis can profoundly disturb skin homeostasis and alter susceptibility to neoplastic development.

Association between high levels of ornithine decarboxylase activity and favorable prognosis in human colorectal carcinoma.

Several studies have documented increased expression of ornithine decarboxylase (ODC) in neoplastic colorectal tissue versus normal-appearing colonic mucosa. The present study was undertaken to determine whether there is an association between the degree of overexpression of ODC in colorectal carcinomas and survival in a series of 74 patients. A high level of tumor ODC expression was found to be significantly associated with greater survival in our patient series. Patients with tumor ODC activities greater than the median and especially in the highest quartile experienced a more favorable outcome than those patients with ODC values below the median or in the lowest quartile (P = 0.03 and 0.02, respectively). The presence of a GTP-activatable isoform of ODC was also significantly associated with a favorable prognosis but only in tumors of the right colon (P = 0.01). There was no association found between ODC activity and tumor grade, tumor size, or patient age, sex, or race. Our results demonstrate that high levels of ODC expression (and presence of a GTP-activatable isoform for right-sided colon tumors) predict a favorable prognosis in human colorectal carcinoma. Knowledge of a patient's ODC status at the time of surgery may be useful in decisions regarding adjuvant therapy. Understanding the mechanism(s) involved should lead to new therapeutic approaches for advanced colorectal carcinoma.

Induction of ornithine decarboxylase in specific subpopulations of murine epidermal cells following multiple exposures to 12-O-tetradecanoylphorbol-13-acetate, mezerein and ethyl phenylpropriolate.

Single applications of 12-O-tetradecanoylphorbol-13-acetate (TPA), mezerein or ethyl phenylpropriolate (EPP) to mouse skin at appropriate doses cause similar degrees of hyperplasia and comparable levels of induction of epidermal ornithine decarboxylase (ODC) activity. Multiple (n = 5) treatments with these agents, in contrast, resulted in large differences in induced ODC activity (TPA much greater than mezerein greater than EPP) with no differences in the degree of hyperplasia or [3H]thymidine pulse-labeling among the multiple treatment groups. To attempt to explain the cellular basis for the greater ODC-inducing ability of TPA relative to mezerein and EPP in chronic exposure protocols, immunocytochemical and flow cytometric analyses were performed. Immunocytochemistry using an ODC-specific polyclonal antibody revealed substantially different pattern of ODC-positive cells in chronically exposed epidermis than observed with single exposures. TPA treatment resulted in very pronounced immunostaining of the perifollicular cells, with little evidence of specific staining in the interfollicular epidermis mezerein treatment yielded staining in both interfollicular and some perifollicular areas, while EPP treatment produced the least amount of specifically stained cells, all of which were in the interfollicular epidermis. Flow cytometric analysis of keratinocytes isolated from chronically treated skin identified three distinct subpopulations that bound varying amounts of ODC antibody. Chronic treatment of CD-1 murine epidermis with TPA appeared to cause the expansion of an intermediate sized cell subpopulation that was not apparent with EPP or mezerein. Our results suggest that chronic treatment of murine epidermis with the potent complete tumor promoter TPA leads to the selective expansion of a keratinocyte subpopulation that is hyperinducible for ODC and may be identical to the cells in the perifollicular region previously identified. These observations also suggest that the weaker tumor promoters mezerein and EPP are less capable of causing expansion of this specific subpopulation, which may be an important target cell population for neoplastic transformation in mouse epidermis.

Identification of epidermal cell subpopulations with increased ornithine decarboxylase activity following treatment of murine epidermis with 12-O-tetradecanoylphorbol-13-acetate.

Ornithine decarboxylase (ODC), the initial enzyme in the polyamine biosynthetic pathway, has been used as a marker for the hyperplasia that occurs following exposure of mouse epidermis to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Using flow cytometry in combination with polyclonal antibodies to ODC, we examined the levels of ODC-associated immunoreactive protein present within mouse epidermal cells at 4 and 24 h after a single topical application of TPA, as well as following chronic exposure to TPA and in papillomas. Basal levels of ODC-specific antibody binding were detectable in acetone-treated CD-1 mouse epidermis and were increased 3-fold at 4 h after TPA treatment. The amount of ODC antibody binding detected after exposure to 17 nmol TPA twice weekly for 3 weeks was similar to that detected within cells isolated from papillomas and was 2.5-fold higher than in cells isolated at 4 h after a single topical treatment of mice with TPA. These observations support the hypothesis that specific subpopulations of keratinocytes constitutively express high levels of ODC following chronic exposure to TPA. The novel method for ODC detection described in these studies provides a means to identify, isolate, and further characterize epidermal cells that may give rise to papillomas and carcinomas.

Properties of ornithine decarboxylase in human colorectal adenocarcinomas.

Ornithine decarboxylase (ODC) activity was measured in colon adenocarcinomas and adjacent normal-appearing colon mucosa from a total of 40 patients undergoing surgical resections. The enzyme activity was measured in the presence and absence of GTP, since recent work has demonstrated a GTP-activatable form of ODC in some murine and human tumors. In general, ODC specific activity was higher in adenocarcinomas than in adjacent normal-appearing mucosa. Of greater interest, however, was the finding that 13 of 40 tumors and 3 of 40 mucosae contained a GTP-activatable form of ODC. These are minimal estimates of the proportion of tissues positive for this enzyme form, since a multiple sampling protocol indicated that expression of a GTP-activatable ODC was not uniform throughout a given tumor. Chromatographic analyses of tumor extracts revealed the presence in some tumors of multiple size forms of ODC, only some of which were activated by GTP. Enzyme kinetic data indicated that the multiple forms of ODC can have different affinities for L-ornithine and that GTP can "normalize" the aberrant kinetic properties of these forms. While there was no statistically significant correlation of the presence of a GTP-activatable ODC with stage of disease, analysis of our data revealed a positive association of a GTP-activatable ODC with tumor site; a much higher percentage of tumors of the cecum contained this ODC isoform than tumors of other colonic segments (64% versus less than or equal to 25% for other sites). These results demonstrate (a) the presence of a functionally distinct form of ODC in some human colon adenocarcinomas and (b) a distinct regional distribution of this ODC form within the colon. We suggest this alteration in a key enzyme in the growth-associated pathway of polyamine biosynthesis may play a role in colon tumor progression.

Flow cytometric detection of ornithine decarboxylase activity in epidermal cell subpopulations.

Using flow cytometry in combination with membrane permeabilization techniques to enhance binding of antibodies with immunoreactive protein within the cytoplasm, we have developed a method to examine the ornithine decarboxylase (ODC) activity present within subpopulations of epidermal cells following acute and chronic exposure to the phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA). The method described has the sensitivity to detect basal levels of ODC as well as increases in ODC at early time points following treatment with TPA and has the additional advantage of allowing subpopulation identification and characterization.

Regulation of ornithine decarboxylase gene expression in normal and transformed hamster embryo fibroblasts following stimulation by 12-O-tetradecanoylphorbol-13-acetate.

We have compared the regulation of ornithine decarboxylase (ODC) gene expression in primary cultures of hamster embryo fibroblasts and in two independently transformed hamster embryo cell lines. Previous studies have demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) can greatly potentiate the serum growth factor induction of ODC enzyme activity in transformed cells, but not in normal hamster embryo fibroblasts. Treatment of either normal or transformed cells with both TPA and serum yielded greater accumulations of ODC mRNA than with either treatment alone, which is consistent with changes at the protein level. However, treatment of the transformed cells with TPA and serum resulted in a greater increase in steady state levels of ODC mRNA than that observed using normal fibroblasts. The time course for the induction of ODC mRNA was similar for both normal and transformed cells with maximal accumulations 4-8 h after treatment. Studies with actinomycin D further suggests that ODC mRNA is comparatively long-lived in both normal and transformed cells. The accumulation of ODC mRNA after stimulation with TPA and serum is blocked by cycloheximide in normal hamster fibroblasts suggesting that this induction is dependent upon protein synthesis. In contrast, cycloheximide did not affect the accumulation of ODC mRNA under similar treatment conditions in transformed cells. This altered regulation of ODC gene expression in transformed hamster embryo fibroblasts cannot be explained by either gene rearrangement or the amplification of an ODC gene. These data suggest that transformation of hamster embryo cells results in a loss of cellular control over ODC gene regulation which includes an alteration in the requirement for protein synthesis for ODC mRNA accumulation.

Activation of human squamous cell carcinoma ornithine decarboxylase activity by guanosine triphosphate.

Previous studies have demonstrated the presence in mouse epidermal tumors of a structurally and functionally altered ornithine decarboxylase (ODC). In this report, the enzymatic properties of ODC from normal human skin and squamous cell carcinomas are examined. Some tumors contained a more heat stable ODC than the enzyme found in normal skin. GTP stimulated enzyme activity in four of seven tumor extracts tested but had no effect on normal skin ODC. Kinetic analyses indicated that GTP either lowered the apparent Km of tumor ODC for L-ornithine, increased the Vmax, or had both effects, depending on the tumor examined. Gel filtration chromatography of crude tumor extracts indicated the existence of multiple molecular weight forms of ODC, some of which can be activated by GTP and some of which are unaffected by GTP. Some tumors contain both a GTP-activatable and -nonactivatable form of the enzyme. Immunolocalization studies demonstrated the presence within squamous cell carcinomas of cells with a constitutively high level of immunoreactive ODC, a situation never observed in normal skin tissue. These results suggest that some human squamous cell carcinomas contain a functionally altered ODC that may be aberrantly regulated.