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[Cancer Research 60, 5696-5703, October 15, 2000]
© 2000 American Association for Cancer Research

Carcinogenesis

Inhibition of Ornithine Decarboxylase (ODC) Decreases Tumor Vascularization and Reverses Spontaneous Tumors in ODC/Ras Transgenic Mice1

Li Lan, Carol Trempus and Susan K. Gilmour2

The Lankenau Institute for Medical Research, Wynnewood, Pennsylvania 19096 [L. L., S. K. G.], and Laboratory of Environmental Carcinogenesis and Mutagenesis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 [C. T.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The polyamines have long been known to be associated with rapid cell proliferation in both normal and neoplastic cells and tissue (1 , 2) . ODC3 is the initial rate-limiting enzyme involved in the biosynthesis of polyamines and is responsible for converting L-ornithine to putrescine. The polyamines putrescine, spermidine, and spermine are some of the major cations present in cells, and they are essential for normal cell growth and differentiation. ODC is aberrantly regulated in tumor cells and results in high basal levels of polyamines in many epithelial tumors (3, 4, 5, 6, 7) . In large part, this results from the up-regulation of ODC expression by oncogenes such as c-myc (8, 9, 10) , v-src (11) , v-raf (12) , or an activated Ras or RhoA protein (12 , 13) . Indeed, the mutation of c-Ha-ras is an early genetic change in mouse skin initiated with the carcinogen DMBA (14) . Although mutations in the ras gene occur in 30% of all human tumors (15) , aberrant Ras signaling probably contributes even more to the development of human tumors because mutations in other genes can also lead to chronic up-regulation of the Ras pathway without any mutations in ras (16, 17, 18) .

To study the effect of elevated levels of ODC and polyamines and a mutant ras gene in mouse skin tumorigenesis, we generated double transgenic mice by breeding K6/ODC transgenic mice with TG.AC v-Ha-ras transgenic mice (19) . A keratin 6 promoter drives the ODC transgene in K6/ODC transgenic mice, which results in elevated ODC/polyamine levels directed to the outer root sheath of hair follicles (20) . TG.AC transgenic mice carry a v-Ha-ras transgene while still retaining two normal c-Ha-ras alleles (21 , 22) . Transgenic mice that possess only the K6/ODC or the v-Ha-ras transgene do not develop skin tumors unless treated with either a carcinogen or tumor promoter, respectively (23, 24, 25) . However, we found that ODC overexpression, targeted to hair follicles in conjunction with an activated Ras protein, are sufficient to produce spontaneous skin carcinomas in ODC/Ras double transgenic mice (19) .

The ODC/Ras transgenic mouse offers several advantages as a model for studying epithelial tumorigenesis, including the ability to focus on the effects of aberrant expression of only two genes (Ras and ODC) without any complicating effects of chemical carcinogens or tumor promoters on other genes. Another advantage is that the K6/ODC transgene expression can be manipulated with the highly specific ODC inhibitor, DFMO (26) . DFMO has been shown to inhibit the development of skin tumors in carcinogen-treated mice when it is given during the promotion phase (27 , 28) , and it is effective in treating glioblastomas when combined with 1,3-bis(2-chloroethyl)-1-nitrosourea (29) . Currently, DFMO is being used with moderate success in cancer chemopreventive trials with patient populations at high risk for the development of colon, esophageal, breast, skin, and prostate cancer (30) .

We report here that treatment of ODC/Ras transgenic mice with 1% DFMO in the drinking water not only prevents the formation of spontaneous skin tumors but also causes the rapid regression of tumors that are allowed to spontaneously develop. Surprisingly, we found that the proliferation index of the tumor epithelial cells in regressed ODC/Ras tumors was not affected by DFMO. Our results suggest that polyamines play an important role in the survival of epidermal tumors, perhaps by controlling essential angiogenic factors.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
ODC/Ras double transgenic mice were generated by crossing K6/ODC transgenic mice on a C57BL/6 background with TG.AC transgenic mice homozygous for the v-Ha-ras transgene and on a FVB/N background, as described previously (19) . Use of animals was approved by the Institutional Animal Care and Use Committee of the Lankenau Institute for Medical Research. Skin tumors spontaneously developed in ODC/Ras transgenic mice hemizygous for both the ODC and the v-Ha-ras transgenes 6–10 weeks after birth. Tumors >1 mm in size were counted and measured with calipers at least twice a week. To delay the formation of spontaneous tumors, 4-week-old mice were administered DFMO (Ilex Oncology, Inc., San Antonio, TX) at 1% (w/v) in the drinking water for 5 weeks. For tumor regression studies, mice with tumors were administered 1% DFMO in the drinking water for up to 6 weeks. Two h before sacrifice, all mice were injected i.p. with bromodeoxyuridine (Sigma, St. Louis, MO) at a dose of 100 µg/g body weight.

Processing of Tissues.
A portion of each tumor was processed for histology, and the remainder was snap frozen in liquid nitrogen. To circumvent problems arising from tumor heterogeneity, frozen tumor tissue was ground to a fine powder and used for subsequent RNA, protein, and polyamine analyses. For ODC enzyme activity, a portion of the ground tissue was lysed in 25 mM Tris-HCl (pH 7.5), 2.5 mM DTT, 0.1 mM EDTA, and protease inhibitors. Homogenates were assayed for ODC enzyme activity by quantifying the production of 14CO2 from L-[14C]ornithine (31) . A portion of ground tissue was also lysed in 0.2 N perchloric acid for measurement of polyamine levels. Putrescine, spermidine, and spermine levels were determined by dansylation and separation on a reversed phase C18 high-performance liquid chromatography column (32) .

Protein Analyses.
For immunoblots, tissues were homogenized in RIPA buffer [50 mM Tris-HCl (pH 7.5), 1% NP40, 0.25% sodium deoxycholate, 0.25% SDS, 150 mM NaCl, and 1 mM EGTA] containing 1 µg/ml each of aprotinin, leupeptin, and pepstatin, 1 mM NaF, 1 mM sodium orthovanadate, and 1 mM Pefabloc by passing through a syringe needle after a 30-min incubation on ice. Tissue lysates were clarified by centrifugation, and protein content was determined by a Lowry assay. Equal amounts of protein were separated by 12% SDS-polyacrylamide gel and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). To assess for equal protein loading and transfer to the membranes, the protein on the membranes was stained with Ponceau S solution (Sigma Chemical Co., St. Louis, MO). Immunoblots were incubated for 1 h at room temperature in blocking solution (PBS with 10% milk and 0.05% Tween 20), followed by the primary antibody diluted in blocking solution containing 0.1% milk for 1–2 h. Blots were probed with a polyclonal anti-keratin 1 or loricrin antibody (Covance, Richmond, CA) and a monoclonal anti-ß-actin antibody (Sigma). The immunoblots were developed with a horseradish peroxidase-conjugated secondary antibody, followed by detection using enhanced chemiluminescence according to the manufacturer’s directions (Amersham Pharmacia Biotech, Inc., Piscataway, NJ).

Immunohistochemistry/in Situ Hybridization.
Tissues were fixed in either Fekete’s solution (60% ethanol, 3.2% formaldehyde, and 0.75 M acetic acid) or in 4% p-formaldehyde in PBS overnight and then embedded in paraffin. Sections were deparaffinized, hydrated, and incubated with primary antibody. Slides were then incubated with the appropriate biotinylated secondary antibody, followed by an incubation with an avidin and biotinylated peroxidase complex (Vectastain Elite ABC kit; Vector Laboratories, Inc., Burlingame, CA), all at room temperature. Immunoreactive cells were localized by incubating the sections with a chromagen solution containing diaminobenzidine and H2O2 and then counterstaining with hematoxylin. Cyclin D1 expression was detected using a rabbit polyclonal anti-cyclin D1 antibody (Upstate Biotechnology, Lake Placid, NY). Keratin 1 and loricrin expression was detected using rabbit polyclonal antibodies obtained from Covance (Richmond, CA). BrdUrd incorporated in cells undergoing DNA synthesis was detected in tumor sections using a rat monoclonal anti-BrdUrd antibody (Zymed Laboratories, San Francisco, CA). The proliferative index was determined by multiplying the number of BrdUrd-positive cells/500 tumor epithelial cells in the basal layer by 100. To visualize the vasculature of tumors, endothelial cells were stained with a monoclonal antimouse CD31 (NeoMarkers, Inc., Union City, CA). Vessels were counted in five areas at x10 (eyepiece) and x10 (objective) as described by Bolontrade et al. (33) . The vessel count for each sample was determined as the average number of vessels from five areas. Serial sections of tumors were also processed for in situ hybridization with 35S-labeled sense or antisense riboprobe to the SV40 polyadenylation/splice sequence of the v-Ha-ras transgene transcript as described previously (19 , 34) .

Apoptosis Analysis.
Freshly sectioned tissue was deparaffinized with xylene, and apoptotic cells were detected using the ApoTACS TdT-based TUNEL assay (Trevigen, Gaithersburg, MD), following the manufacturer’s instructions. Briefly, tumor sections were deparaffinized with xylene and then rehydrated and permeabilized with proteinase K in PBS for 30 min. After washing the sections, DNA fragments in apoptotic cells were end-labeled with biotinylated nucleotides using a TdT/horseradish peroxidase reaction in conjunction with an antibody detection system targeting nucleotides incorporated onto the 3'-OH ends of the DNA fragments. DNA fragmentation was detected by staining with diaminobenzidine, and cells were counterstained with methyl green. Positive control tissue sections were prepared by nicking DNA with DNase I. In negative control tissue sections, deionized water was substituted for TdT in the working solution. Apoptotic cells were identified on the basis of a combination of positive staining and morphological criteria as described by Kerr et al. (35) . The apoptotic index was determined by dividing the number of apoptotic cells by the total number of cells in the tumor and multiplying by 100.

RT-PCR.
RNA was isolated from frozen tumor tissue using Tri Reagent (Molecular Research Center, Inc., Cincinnati, OH). cDNA was synthesized by incubating RNA with oligo dTs and reverse transcriptase at 42°C for 1 h. Amplification of VEGF120, VEGF164, VEGF188, and HPRT message was carried out essentially as described by Tober et al. (36) . Briefly, the cDNA was amplified by an initial denaturation at 95°C for 2.5 min and then 30 cycles of PCR (95°C for 15 s, 62°C for 30 s, and 72°C for 30 s), followed by a 7-min 72°C final elongation step. Products of PCR amplification were analyzed on a 2% agarose gel.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We have shown previously that transgenic mice expressing both the K6/ODC and v-Ha-ras transgenes (ODC/Ras transgenic mice) develop spontaneous skin tumors early in life. The development of these tumors is dependent upon elevated levels of ODC enzyme activity because tumor formation can be blocked if mice are administered 1% DFMO in their drinking water (19 , 25 , 37) . No skin tumors developed in littermates that were hemizygous for the v-Ha-ras transgene but lacked the ODC transgene. Removal of the DFMO from the drinking water resulted in formation of spontaneous tumors in ODC/Ras mice approximately 4–7 weeks later (Fig. 1)Citation . The rate of tumor incidence is similar, regardless of whether tumor formation was delayed with prior DFMO treatment, thus reflecting that spontaneous tumors are initially observed in ODC/Ras transgenic mice within a very narrow window of time. On average, fewer skin tumors developed in mice that had been treated previously with DFMO for 5 weeks (mean, 5.5 tumors/mouse) compared with the number of tumors that developed in mice never treated with DFMO (mean, 12 tumors/mouse).



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  		Fig. 1. ODC/Ras double transgenic mice develop spontaneous skin tumors in a short time span that is dependent on ODC activity. Heterozygous K6/ODC transgenic mice were bred with homozygous TG. AC v-Ha-ras transgenic mice and some pups were given 1% DFMO in the drinking water for 5 weeks starting at the age of 4 weeks. ODC/Ras double transgenic offspring in the two groups were observed for the appearance of spontaneous skin tumors, and the percentage of tumor incidence is shown as the number of mice with tumors at a given age/total number of mice in the group.

 
DFMO treatment of ODC/Ras transgenic mice caused a rapid regression of the tumors that had developed spontaneously (Fig. 2)Citation . Tumors >1 cm2 almost completely regressed within 2–4 weeks of DFMO treatment, with no apparent toxicity to the animals. Table 1Citation shows that most tumors regressed quite dramatically in the first week or two after administration of DFMO. Interestingly, histological examination revealed that the tumor epithelial cells that remained in the regressing tumors never completely disappeared with DFMO treatment. Despite almost complete visible regression of the tumors after 6 weeks of DFMO treatment, small clusters of hyperplastic growths of epithelial cells were still present upon histological evaluation of dermal tissue where tumors had been seen previously (data not shown). Indeed, cessation of DFMO treatment resulted in the regrowth of some of the regressed tumors.



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  		Fig. 2. Regression of ODC/Ras tumors with DFMO treatment. The same ODC/Ras double transgenic mouse (13 weeks of age on day 1 of DFMO treatment) is shown: A, no DFMO treatment; B, 4 days DFMO; C, 12 days DFMO; D, 42 days DFMO.

 

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  		Table 1 DFMO-induced regression of spontaneous tumors in ODC/Ras double transgenic mice

 
Expression of ODC and v-Ha-ras Transgenes in DFMO-regressed Tumors.
ODC enzyme activity was dramatically decreased both in the regressed tumors and the adjacent, non-tumor-bearing skin after 1 day of DFMO treatment (Table 2)Citation . As expected, the decrease in putrescine levels mirrored the drop in ODC activity observed in both skin and tumors within the first day of DFMO treatment. However, there was no significant change in spermidine or spermine levels in the regressed tumors after 12 days of DFMO treatment, despite the precipitous decline in both ODC activity and putrescine levels (data not shown).


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  		Table 2 Effect of DFMO on ODC activity and putrescine levels in skin and tumors

 
We were particularly interested in whether the v-Ha-ras transgene was still expressed in the regressed tumors. By in situ hybridization, we have detected v-Ha-ras transgene mRNA in ODC/Ras spontaneous tumors with the strongest signal in the basal epidermoid cells of the tumors (19) . After 1 month of DFMO treatment, v-Ha-ras mRNA was still detected in the remaining basal epithelial cells and some suprabasal cells of all DFMO-regressing tumors (Fig. 3)Citation . In addition, elevated levels of cyclin D1 protein were detected in the same basal tumor cells of regressed tumors that expressed v-Ha-ras mRNA. These findings agree with previous reports that cyclin D1 is overexpressed in tumors known to have a mutated ras gene (38, 39, 40, 41) . It has been postulated that cyclin D1 is a downstream mediator of an activated Ha-Ras (42) .



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  		Fig. 3. Cyclin D1 and v-Ha-ras expression in DFMO-regressed ODC/Ras tumors. Serial sections were made from the same regressing tumor (B and D are views at a higher magnification) taken from an ODC/Ras double transgenic mouse treated 12 days with DFMO. Some sections were immunohistochemically stained using an anti-cyclin D1 antibody (A and B). In situ hybridization to detect the v-Ha-ras transgene message was performed on serial sections using a 35S-labeled antisense riboprobe to the SV40 polyadenylation/splice sequence of the v-Ha-ras transgene construct (C and D). Similar levels of expression of cyclin D1 and v-Ha-ras mRNA were also seen in tumors from ODC/Ras mice not treated with DFMO. No signal was detected in serial sections of tumors using the 35S-labeled sense riboprobe. Scale bars: A and C, 100 µm; B and D, 50 µm.

 
Effect of DFMO on Proliferation and Differentiation in Regressed Tumors.
Because elevated cyclin D1 is associated with actively cycling cells (43, 44, 45) , we examined the proliferative index of the epithelial cells in regressed tumors compared with ODC/Ras tumors from mice not given DFMO. Table 3Citation shows that the tumor cell proliferative index was not affected because the majority of basal cells in the tumor continued to incorporate BrdUrd, as indicated by numerous BrdUrd-stained nuclei in the basal cells of regressed tumors after 42 days of DFMO treatment. These data imply that the major contribution of elevated levels of ODC/polyamines in cooperating with a mutated ras to produce tumors is some process other than increased tumor cell proliferation or sustained cyclin D1 expression. Because there was no effect on the proliferative index of basal epithelial cells in the DFMO regressed tumors, increased cell death, through either terminal differentiation or apoptosis, must account for the decrease in tumor mass after inhibition of ODC enzyme activity.


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  		Table 3 Effect of DFMO on proliferative and apoptotic index in ODC/Ras tumors

 
Immunohistochemical staining of tumors detected no increased expression of differentiation markers such as keratin 1 or loricrin in regressing tumors as compared with tumors from mice not treated with DFMO (Fig. 4a)Citation . Immunoblot analyses revealed that keratin 1 expression is elevated in the epidermis of K6/ODC transgenic mice (Fig. 4b)Citation . Expression of keratin 1 appears to be dependent upon polyamine levels because 4 days of DFMO treatment lowered the level of keratin 1 protein in the epidermis of both K6/ODC transgenic mice and their normal littermates (Fig. 4b)Citation . Although an activated Ha-ras has been shown to block the expression of keratin 1 in epidermal cells and tumors (46 , 47) , some keratin 1 protein was still expressed in the spontaneous ODC/Ras tumors (Fig. 4)Citation . Interestingly, inhibiting ODC enzyme activity via treatment with DFMO resulted in less keratin 1 expression in the regressed tumors (Fig. 4)Citation . In addition, immunoblot analysis has not revealed increased expression of loricrin, another epidermal differentiation marker, in DFMO regressed tumors compared with spontaneous tumors from ODC/Ras transgenic mice not treated with DFMO (data not shown). Although the accumulation of keratin 1 and loricrin is not elevated in DFMO regressed tumors, it is likely that changes in the rate of differentiation will not be detected if these differentiation products are being sloughed off at an increased rate as well. However, the lack of evidence of any increased expression of these differentiation markers in regressing tumors suggests that regression of ODC/Ras tumors does not appear to be attributable to accelerated terminal differentiation of the tumor epithelial cells.



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  		Fig. 4. Detection of differentiation and apoptotic cells markers in regressed and nonregressed skin tumors from ODC/Ras transgenic mice. a, serial sections of a tumor from an ODC/Ras transgenic mouse not treated with DFMO (A, B, and E) and of a regressing tumor from an ODC/Ras mouse treated with DFMO for 4 days (C, D, and F). Sections were immunohistochemically stained using an anti-keratin 1 antibody (A and C) or an anti-loricrin antibody (B and D). TUNEL analysis of a nonregressed tumor (E) and a regressed tumor (F) revealed dark brown-stained positive cells in F, some of which are indicated by the arrows. Scale bars: 50 µm. b, immunoblot analysis of the effect of DFMO on keratin 1 expression in skin and tumors from K6/ODC and ODC/Ras transgenic mice. RIPA lysates of epidermal (E) and dermal (D) tissue from K6/ODC mouse skin (K6/ODC) and nontransgenic littermate (WT) mouse skin and of tumors from ODC/Ras double transgenic mice were prepared after treatment for 0, 1, 4, or 12 days with 1% DFMO in the drinking water. Equal amounts of protein were loaded and resolved by SDS-PAGE and transferred to nitrocellulose membranes. Keratin 1 and ß-actin protein were detected by immunoblotting.

 
DFMO Treatment Increases the Apoptotic Index in ODC/Ras Regressed Tumors.
TUNEL analyses revealed increased DNA fragmentation in DFMO regressing tumors compared with similarly sized spontaneous tumors from ODC/Ras transgenic mice not treated with DFMO (Fig. 4aCitation ; Table 3Citation ). Apoptotic cells were detected in both the tumor epithelial cells as well as the stromal cells of regressing tumors. It was necessary to compare similarly sized tumors because large ODC/Ras tumors often presented with a considerable degree of apoptosis in tumor epithelial cells found at the distal ends of the tumor fronds, most likely because of an inadequate blood supply in these rapidly growing keratoacanthomas. The regressed tumors, as well as the ODC/Ras tumors that were of similar small size from mice never given DFMO, did not demonstrate this same high degree of apoptotic cells at the tips of the tumor fronds. In addition, TUNEL-stained stromal cells were never detected in tumors from ODC/Ras mice not treated with DFMO but were detected only in the DFMO regressing tumors. As confirmation of these results, we also used an independent marker of apoptosis, caspase-3. Immunoblot analyses revealed caspase-3-stained cells in DFMO-regressed tumors but not in similarly sized tumors from ODC/Ras transgenic mice not treated with DFMO (data not shown).

Decreased Vasculature in the Regressed Tumors.
Because a good blood supply is necessary for tumor growth and the maintenance of tumor tissue, we looked for evidence of effects of decreased ODC activity on the tumor vasculature in DFMO-regressed tumors. Upon visible inspection of the tumors, it was apparent that tumors appeared more pale and less vascularized within the first four days of DFMO treatment, even before the tumor size became reduced to any large extent (Fig. 5)Citation . Table 4Citation shows that the blood vessel count was significantly decreased in regressed tumors after 1 and 4 days of DFMO treatment. Indeed, we have consistently observed increased vasculature in the dermis of K6/ODC skin compared with dermis from nontransgenic littermates (Table 4)Citation . These results suggest that elevated ODC enzyme activity contributes to the survival of these epidermal tumors by up-regulating essential angiogenic factors.



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  		Fig. 5. Inhibition of ODC activity decreases the tumor vascularization. Two ODC/Ras transgenic mice with tumors (A and C) are pictured before any treatment with DFMO. After treatment with DFMO for 4 days, tumors shown in A at 0 days DFMO are now shown in B, and tumors shown in C at 0 days DFMO are now shown in D. Note the decreased vasculature in the tumors (arrows) after 4 days of DFMO treatment (B and D).

 

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  		Table 4 Effect of ODC overexpression on the vasculature in transgenic mouse skin and tumors

 
Because VEGF is known to be a potent mediator of vasculogenic and angiogenic events associated with tumor growth (48, 49, 50, 51, 52) , we looked at the effect of DFMO inhibition on VEGF expression in the regressed tumors. Similar to the human VEGF gene, the mouse gene for VEGF has been shown to encode three alternatively spliced forms of VEGF (VEGF188, VEGF164, and VEGF120) but no isoform corresponding to the human VEGF206 (53) . RT-PCR analysis of mRNA isolated from ODC/Ras tumors from transgenic mice not treated with DFMO, or from regressed tumors from mice treated 1, 4, or 12 days with DFMO, revealed no change in the expression level of VEGF120, VEGF164, or VEGF188 (Fig. 6)Citation . Moreover, Western analysis showed no increased expression of VEGF protein in K6/ODC transgenic skin compared with normal littermate skin, despite the increased vasculature in K6/ODC skin (data not shown). However, VEGF protein remained elevated in spontaneous tumors from ODC/Ras transgenic mice. VEGF mRNA up-regulation has been shown to correlate with activated Ha-ras levels (54) , and our results showing no change in the level of VEGF isoforms in regressed ODC/Ras tumors reflects that v-Ha-ras expression is not affected in the DFMO regressed tumors. Thus, our findings suggest that the decreased vasculature in the regressed tumors is attributable to altered activity of an essential angiogenic factor(s) other than VEGF.



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  		Fig. 6. Semiquantitative RT-PCR shows no change in the expression of murine VEGF isoforms in regressed ODC/Ras tumors after DFMO treatment. Total RNA was isolated from skin tumors of ODC/Ras transgenic mice after 0, 1, 4, or 12 days of DFMO treatment. Equal amounts of mRNA as assessed by RT-PCR with primers specific for the housekeeping gene HPRT (lower panel) were subjected to RT-PCR with primers specific for VEGF120, VEGF164, and VEGF188 transcripts. VEGF and HPRT cDNA products are shown on ethidium bromide-stained 2% agarose gels.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
More than 90% of skin tumors induced by the standard DMBA and TPA tumorigenesis protocol possess a mutation at codon 61 of the Ha-ras gene (14 , 55) , resulting in the production of a constitutively activated Ras protein. Although a mutated Ha-ras oncogene plays an important role in the initiation step of mouse skin carcinogenesis (14 , 55 , 56) , our data indicate that tumor development and progression to a malignant phenotype are also dependent upon elevated levels of ODC and polyamines. In the ODC/Ras transgenic mouse model, an activated ras is still expressed in regressed tumors after inhibition of tumor ODC enzyme activity by DFMO. Although DFMO treatment results in a dramatic reduction in tumor size, the continued expression of v-Ha-ras presumably permits the observed continued proliferation and up-regulation of cyclin D1 in epithelial tumor cells (42, 43, 44, 45) .

These seemingly paradoxical results of tumor regression despite continued proliferation were initially surprising in light of the strong association of ODC activity induction with proliferating cells and tissue (1 , 2 , 57 , 58) . Moreover, we have demonstrated previously that sustained elevated levels of ODC and polyamines stimulate proliferation in the skin of K6/ODC transgenic mice compared with that in normal littermate skin (59) . Indeed, our results showing no inhibitory effect of DFMO on DNA synthesis in regressing ODC/Ras tumors differ from those reported by O’Brien et al. (37) , who examined the effect of DFMO on DMBA-initiated tumors in K6/ODC transgenic mice and found a dramatic decrease in tumor cell proliferation in regressed tumors. Because these were carcinogen-induced tumors, it is important to note that carcinogens, including DMBA, target many genes other than Ha-ras, which may exert different effects on cell cycle progression (60) . Moreover, there may be a ras dosage effect because ODC/Ras transgenic mice carry a v-Ha-ras transgene, having point mutations in codons 12 and 59 in addition to two normal c-Ha-ras alleles. Other factors contributing to differences in DFMO-induced regression of ODC/Ras tumors and DMBA-initiated K6/ODC tumors may lie in the different etiology of the tumors or in the genetic background of the mice. In fact, all of the spontaneous tumors that develop in ODC/Ras double transgenic mice are keratoacanthomas, unlike the squamous papillomas that form in the DMBA-initiated K6/ODC transgenic mice, which may reflect the FVB/N genetic background in the ODC/Ras double transgenic mice. In contrast to its human counterpart, keratoacanthomas in mice often convert to squamous carcinomas and usually do not regress (61) . Thus, it is significant that these aggressive keratoacanthomas in ODC/Ras mice regress so quickly with DFMO treatment. However, our results illustrate that elevated levels of polyamines not only affect the proliferation of tumor cells but also promote tumorigenesis and the survival of tumors through downstream effectors that control pathways other than proliferation.

Because DFMO-induced regression of spontaneous tumors in the ODC/Ras transgenic mice does not involve altered expression of v-Ha-ras or changes in the proliferative index of the remaining epithelial tumor cells, then ODC and polyamines must regulate other pathways leading to either terminal differentiation or apoptosis that would yield a net decrease in a proliferating population of tumor cells. One possible explanation for the regression of ODC/Ras tumors promoted by DFMO treatment is that DFMO may block one of the effector pathways of activated Ha-Ras (62) that determines cell survival. For instance, it has been reported that the phosphatidylinositol 3-kinase pathway, acting through PKB/Akt, mediates the aberrant survival of Ras-transformed epithelial cells in the absence of attachment (63) . If polyamines play a key role in this survival pathway, then it would be expected that DFMO treatment would act to abrogate this protection against apoptosis, thus resulting in tumor regression.

Our data suggest that differentiation of tumor epithelial cells is not accelerated in the regressing tumors because there is no increase in the expression of genes normally associated with the terminal differentiation program of keratinocytes. However, there is a positive correlation of DFMO-induced regression of ODC/Ras tumors with decreased vascularization and increased apoptosis of both tumor epithelial and stromal cells. It remains to be determined what essential survival factors and/or angiogenic factors are regulated by polyamines that play a key role in the maintenance of these tumors. This pattern of increased apoptosis and decreased tumor vascularization in regressing tumors is similar to that observed upon treatment of primary tumors with angiogenesis inhibitors such as angiostatin and endostatin (64 , 65) . Indeed, angiogenesis is essential for the growth and persistence of solid tumors (66, 67, 68) , and it is possible that DFMO inhibition of ODC activity and the resultant decreased vascularization of tumors leads to a critical reduction of paracrine factors essential for the survival of tumor cells. Accumulating reports indicate that polyamines play an important role in tumor neovascularization. DFMO has been found to inhibit B16 melanoma-induced angiogenesis in chick embryo chorioallantoic membranes (69) and to affect the first step in the metastatic cascade, intravasation (70) . In addition, ODC-transformed NIH3T3 cells produce well-vascularized, rapidly growing fibrosarcomas (71 , 72) .

Neovascularization is the result of the net balance between positive and negative regulators of this process. Not only do tumors up-regulate the production of a variety of angiogenic factors such as VEGF, acidic FGF, and basic FGF (54 , 73) , they also generate inhibitors of angiogenesis including angiostatin (64) , thrombospondin (74) , and endostatin (65) . Expression of well-known angiogenic factors such as VEGF, basic and acidic FGFs, or matrix metalloproteinases has not been found to be stimulated by elevated levels of ODC (71) . VEGF is a potent angiogenic factor that is unique in that it acts specifically on vascular endothelial cells (75 , 76) , and different VEGF isoforms have been reported to be selectively expressed in tumors and various tissues of TG.AC transgenic mice, thus suggesting different functions for these various VEGF isoforms (36) . However, we found that DFMO had no effect on the expression of murine VEGF isoforms in regressing ODC/Ras tumors. These findings are expected because they agree with published reports that describe the proliferative effects and the up-regulation of VEGF expression as important downstream effectors of an activated ras oncogene in contributing to tumor growth (54 , 77) . However, we have demonstrated that the proliferative effects of an activated Ha-ras and continued expression of VEGF are not sufficient to support the continued development and survival of these epidermal tumors.

The effect of ODC on the vascularization of tumors is attributable, at least in part, to the production of a yet unidentified angiogenic factor(s). Indeed, our preliminary studies have shown that primary cultures of ODC-overexpressing keratinocytes from K6/ODC transgenic skin attract significantly more endothelial cells to migrate across a Matrigel-coated filter than do normal primary keratinocytes when normal or K6/ODC keratinocytes are cultured in serum-free medium. Because polyamines added to the culture medium of normal keratinocytes are not sufficient to increase the migration of endothelial cells, it is likely that ODC-overexpressing keratinocytes produce a factor that stimulates the migration of endothelial cells. These findings agree with previous reports that conditioned medium from ODC-overexpressing fibroblasts promotes the migration of bovine capillary endothelial cells in collagen gels and increases the growth rate of these endothelial cells in vitro (71) . Although the identity of ODC-induced angiogenic factor(s) that are responsible for tumor vascularization remains elusive, both the angiogenesis-inhibiting factors, thrombospondin-1 and -2, were found to be down-regulated in ODC-transformed NIH3T3 cells (71) . We have found expression of thrombospondin-2 but not thrombospondin-1 to be decreased in primary keratinocytes isolated from K6/ODC transgenic mice as compared with keratinocytes from normal littermate skin (data not shown). Because the more rapid wound healing in the skin of thrombospondin-2 null mice is at least partially attributed to increased angiogenesis in early wounds (78) and elevated levels of ODC and polyamines are elevated during wound repair, we are currently investigating the possibility that polyamines regulate the expression of thrombospondin-2 and other angiogenesis-related genes in skin tumorigenesis.

Very high levels of polyamines are synthesized in ODC overexpressing epithelial cells (72) , and these small polycationic molecules are easily transported out of the cells into the extracellular matrix from which they can be taken up by nearby stromal cells. As a possible consequence, nearby dermal fibroblasts and endothelial cells might become activated by polyamines generated by ODC-overexpressing tumor epithelial cells, thus influencing endothelial cell proliferation and migration. In fact, the relative contributions of factors that regulate the nonneoplastic stromal and tumor epithelial components in tumor development are poorly understood (79 , 80) . Indeed, the whole tumor microenvironment must be considered to understand the role of polyamines in tumorigenesis. Our results indicate that DFMO-induced tumor regression in ODC/Ras transgenic mice is a good model for studying effects of elevated levels of polyamines independent of those on proliferation that play a key role in epithelial tumorigenesis. Furthermore, our work has implications relevant to human cancer because elevated levels of ODC and polyamines in many epithelial tumors are generally believed to be associated with increased proliferation of the tumor cells. Despite the continued expression of an activated Ras accompanied by no decrease in the proliferative index in the tumors, ODC and polyamines clearly appear to affect cell survival pathways that are independent of a Ras-induced proliferation pathway. Although DFMO is being used as a chemopreventive agent, the availability of polyamines from dietary sources appears to undermine the efficacy of this inhibitor. Future studies aimed at identifying key downstream molecules and signaling events that mediate the tumorigenic effects of elevated intracellular levels of polyamines will no doubt lead to more effective chemotherapeutic strategies for treating and preventing cancer.


   ACKNOWLEDGMENTS
 
We thank Mary K. Smith, Mary Goral, and Barry Paul for expert care and treatment of the transgenic mice. We thank our colleagues, Drs. Cheryl Hobbs, Thomas G. O’Brien, and Rebecca Morris for helpful discussions and critical reading of the manuscript. We gratefully acknowledge John Nielands for help with photography and Loretta Rossino and Patricia McElroy for providing expert editorial assistance.


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

1 Supported in part by Grant CA70739 from the National Cancer Institute. Back

2 To whom requests for reprints should be addressed, at The Lankenau Institute for Medical Research, 100 Lancaster Ave., Wynnewood, PA 19096. Phone: (610) 645-8429; Fax: (610) 645-2205; E-mail: gilmours{at}mlhs.org Back

3 The abbreviations used are: ODC, ornithine decarboxylase; DFMO, {alpha}-difluoromethylornithine; BrdUrd, bromodeoxyuridine; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling; TdT, terminal deoxynucleotidyl transferase; DMBA, 7,12-dimethylbenz[a]anthracene; RT-PCR, reverse transcription-PCR; HPRT, hypoxanthine phosphoribosyltransferase; VEGF, vascular endothelial growth factor; FGF, fibroblast growth factor. Back

Received 3/23/00. Accepted 8/25/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Pegg A. E. Recent advances in the biochemistry of polyamines in eukaryotes. Biochem. J., 234: 249-262, 1986.[Medline]
  2. Tabor, C. W., and Tabor, H. Polyamines. Annu. Rev. Biochem., 53. 749–790, 1984.
  3. Pegg A. E. Polyamine metabolism and its importance in neoplastic growth as a target for chemotherapy. Cancer Res., 48: 759-774, 1988.[Abstract/Free Full Text]
  4. O’Brien T. G. The induction of ornithine decarboxylase as an early, possibly obligatory event in mouse skin carcinogenesis. Cancer Res., 36: 2644-2653, 1976.[Medline]
  5. Gilmour S. K., Aglow E., O’Brien T. G. Heterogeneity of ornithine decarboxylase expression in 12-O-tetradecanoylphorbol-13-acetate-treated mouse skin and in epidermal tumors. Carcinogenesis (Lond.), 7: 943-947, 1986.[Abstract/Free Full Text]
  6. Gilmour S. K., Verma A. K., Madara T., O’Brien T. Regulation of ornithine decarboxylase gene expression in mouse epidermis and epidermal tumors during two-stage tumorigenesis. Cancer Res., 47: 1221-1225, 1987.[Abstract/Free Full Text]
  7. Koza R. A., Megosh L. C., Palmieri M., O’Brien T. Constitutively elevated levels of ornithine and polyamines in mouse epidermal papillomas. Carcinogenesis (Lond.), 12: 1619-1625, 1991.[Abstract/Free Full Text]
  8. Bello-Fernandez C., Packham G., Cleveland J. L. The ornithine decarboxylase gene is a transcriptional target of c-Myc. Proc. Natl. Acad. Sci. USA, 90: 7804-7808, 1993.[Abstract/Free Full Text]
  9. Packham G., Cleveland J. L. The role of ornithine decarboxylase in c-Myc-induced apoptosis. Curr. Top. Microbiol. Immunol., 194: 283-290, 1995.[Medline]
  10. Ben-Yosef T., Yanuka O., Halle D., Benvenisty N. Involvement of Myc targets in c-myc and N-myc induced human tumors. Oncogene, 17: 165-171, 1998.[Medline]
  11. Höltta E., Auvinen M., Andersson L. C. Polyamines are essential for cell transformation by pp60v-src delineation of molecular events relevant for the transformed phenotype. J. Cell. Biol., 122: 903-914, 1993.[Abstract/Free Full Text]
  12. Shantz L., Pegg A. E. Ornithine decarboxylase induction in transformation by H-Ras and RhoA. Cancer Res., 58: 2748-2753, 1998.[Abstract/Free Full Text]
  13. Höltta E., Sistonen L., Alitalo K. The mechanisms of ornithine decarboxylase deregulation in c-Ha-ras oncogene-transformed NIH 3T3 cells. J. Biol. Chem., 263: 4500-4507, 1988.[Abstract/Free Full Text]
  14. Balmain A., Brown K., Akhurst R. K., Fee F. M. Molecular analysis of chemical carcinogenesis in the skin. Cell, 46: 72-75, 1988.
  15. Bos J. L. Ras oncogenes in human cancer: a review. Cancer Res., 49: 4682-4689, 1987.[Abstract/Free Full Text]
  16. Ben-Levy R., Paterson H. F., Marshall C. J., Yarden Y. A single autophosphorylation site confers oncogenicity to the Neu/ErbB-2 receptor and enables coupling to the MAP kinase pathway. EMBO J., 13: 3302-3311, 1994.[Medline]
  17. Slamon D. J., Clark G. M., Wong S. G., Levin W. J., Ullrich A., McGuire W. L. Human breast cancer: correlation of relapse and survival with amplification of the Her-2/neu oncogene. Science (Washington DC), 235: 177-182, 1987.[Abstract/Free Full Text]
  18. DeClue J. E., Papageorge A. G., Fletcher J. A., Diehl S., Ratner N., Vass W. C., Lowy D. L. Abnormal regulation of mammalian p21ras contributes to malignant tumor growth in von Recklinghausen (type 1) neurofibromatosis. Cell, 69: 265-273, 1992.[Medline]
  19. Smith M. K., Trempus C. S., Gilmour S. Co-operation between follicular ornithine decarboxylase and v-Ha-ras induces spontaneous papillomas and malignant conversion in transgenic skin. Carcinogenesis (Lond.), 19: 1409-1415, 1998.[Abstract/Free Full Text]
  20. Megosh L., Gilmour S. K., Rosson D., Peralta Soler A., Blessing M., Sawicki J. A., O’Brien T. G. Increased frequency of spontaneous skin tumors in transgenic mice which overexpress ornithine decarboxylase. Cancer Res., 55: 4205-4209, 1995.[Abstract/Free Full Text]
  21. Leder A., Kuo A., Cardiff R. D., Sinn E., Leder P. v-Ha-ras transgene abrogates the initiation step in mouse skin tumorigenesis: effects of phorbol esters and retinoic acid. Proc. Natl. Acad. Sci. USA, 87: 9178-9182, 1990.[Abstract/Free Full Text]
  22. Hansen L. A., Trempus C. S., Mahler J. F., Tennant R. W. Association of tumor development with increased cellular proliferation and transgene overexpression, but not c-Ha-ras mutations, in v-Ha-ras transgenic Tg. AC mice. Carcinogenesis (Lond.), 17: 1825-1833, 1996.[Abstract/Free Full Text]
  23. Spalding J. W., Momma J., Elwell M. R., Tennant R. W. Chemically induced skin carcinogenesis in a transgenic mouse line (TG. AC) carrying a v-Ha-ras gene. Carcinogenesis (Lond.), 14: 1335-1341, 1995.
  24. Hansen L. A., Tennant R. W. Follicular origin of epidermal papillomas in v-Ha-ras transgenic TG. AC mouse skin. Proc. Natl. Acad. Sci. USA, 91: 7822-7826, 1994.[Abstract/Free Full Text]
  25. O’Brien T. G., Megosh L. C., Gilliard G. , and Peralta Soler, A. Ornithine decarboxylase overexpression is a sufficient condition for tumor promotion in mouse skin. Cancer Res., 57: 2630-2637, 1997.[Abstract/Free Full Text]
  26. Metcalf R., Bey P., Danzin C., Jung M. J., Casara P., Vevert J. P. Catalytic irreversible inhibition of mammalian ornithine decarboxylase (E. C.4.1.1.17) by substrate and produce analogues. J. Am. Chem. Soc., 100: 2551-2553, 1978.
  27. Weeks C. E., Hermann A. L., Nelson F. R., Slaga T. S. {alpha}-Difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, inhibits tumor promoter-induced polyamine accumulation and carcinogenesis in mouse skin. Proc. Natl. Acad. Sci. USA, 79: 6028-6032, 1982.[Abstract/Free Full Text]
  28. Takigawa M., Verma A. K., Simsiman R. C., Boutwell R. K. Inhibition of mouse skin tumor promotion and of promoter-stimulated epidermal polyamine biosynthesis by {alpha}-difluoromethylornithine. Cancer Res., 43: 3732-3738, 1983.[Abstract/Free Full Text]
  29. Prados M., Rodriquez L., Chamberlain M., Silver P., Levin V. Treatment of recurrent gliomas with 1,3-bis(2-chloroethyl-1-nitrosourea and {alpha}-difluoromethylornithine. Neurosurgery, 24: 806-809, 1989.[Medline]
  30. Meyskens F. L., Gerner E. W. Development of {alpha}-difluoromethylornithine (DFMO) as a chemoprevention agent. Clin. Cancer Res., 5: 945-951, 1999.[Abstract/Free Full Text]
  31. O’Brien T. G., Simsiman R. C., Boutwell R. K. Induction of the polyamine biosynthetic enzymes in mouse epidermis by tumor promoting agents. Cancer Res., 35: 662-1670, 1975.
  32. Shore L. J., Peralta Soler A., Gilmour S. Ornithine decarboxylase expression leads to translocation and activation of protein kinase CK2 in vivo.. J. Biol. Chem., 272: 12536-12543, 1997.[Abstract/Free Full Text]
  33. Bolontrade M. F., Stern M. C., Binder R. L., Zenklusen J. C., Gimenez-Conti C. J. Angiogenesis is an early event in the development of chemically induced skin tumors. Carcinogenesis (Lond.), 19: 2107-2113, 1998.[Abstract/Free Full Text]
  34. Hansen L. A., Tennant R. Focal transgene expression associated with papilloma development in v-Ha-ras-transgenic TG. AC mice. Mol. Carcinog., 9: 143-154, 1994.[Medline]
  35. Kerr J. F. K., Gobe G. C., Winterford C. M., Harmon B. V. Anatomical methods in cell death. Methods Cell. Biol., 46: 1-27, 1995.[Medline]
  36. Tober K. L., Cannon R. E., Spalding J. W., Oberyszyn T. M., Parrett M. L., Rackoff A. I., Oberyszyn A. S., Tennant R. W., Robertson F. M. Comparative expression of novel vascular endothelial growth factor/vascular permeability factor transcripts in skin, papillomas, and carcinomas of v-Ha-ras Tg. AC transgenic mice and FVB/N mice. Biochem. Biophys. Res. Commun., 247: 644-653, 1998.[Medline]
  37. Peralta Soler A., Gilliard G., Megosh L., George K., O’Brien T. G. Polyamines regulate expression of the neoplastic phenotype in mouse skin. Cancer Res., 58: 1654-1659, 1998.[Abstract/Free Full Text]
  38. Filmus J., Robles A. I., Shi W., Wong M. J., Golombo L. L., Conti C. Induction of cyclin D1 overexpression by activated ras. Oncogene, 9: 3627-3633, 1994.[Medline]
  39. Robles, A. I., and Conti, C. Early overexpression of cyclin D1 protein in mouse skin carcinogenesis. Carcinogenesis (Lond.), 16: 81–786, 1995.
  40. Peters, G. The D-type cyclins and their role in tumorigenesis. J. Cell Sci., S18: 89–96, 1994.
  41. Hung, W. C., Chai, C. Y., Huang, J. S., and Chuang, C. Y. Expression of cyclin D1 and c-Ki-ras gene product in human epithelial ovarian tumors. Hum. Pathol., 27: 1324–1328, 1996.
  42. Robles A. I., Rodriguez-Puebla M. L., Glick A. B., Trempus C., Hansen L., Sicinski P., Tennant R. W., Weinberg R. A., Yuspa S. H., Conti C. J. Reduced skin tumor development in cyclin D1-deficient mice highlights the oncogene ras pathway in vivo. Genes Dev., 12: 2469-2474, 1998.[Abstract/Free Full Text]
  43. Robles A. I., Larcher F., Whalin R. B., Murillas R., Richie E., Gimenez-Conti I. B., Jorcano J. L., Conti C. J. Expression of cyclin D1 in epithelial tissues of transgenic mice results in epidermal hyperproliferation and severe thymic hyperplasia. Proc. Natl. Acad. Sci. USA, 93: 7634-7638, 1996.[Abstract/Free Full Text]
  44. Zwijsen R. M. L., Klompmaker R., Wientjens E. B. J. G. M., Kristel P. M. P., Van Der Burg B., Michalides R. J. A. M. Cyclin D1 triggers autonomous growth of breast cancer cells by governing cell cycle exit. Mol. Cell. Biol., 16: 2554-2560, 1996.[Abstract]
  45. Aktas H., Cai H., Cooper G. M. Ras links growth factor signaling to the cell cycle machinery via regulation of cyclin D1 and the Cdk inhibitor p27KIP1. Mol. Cell. Biol., 17: 3850-3857, 1997.[Abstract]
  46. Huitfeldt H. S., Heyden A., Clausen O. P. F., Thane E. V., Roop D., Yuspa S. H. Altered regulation of growth and expression of differentiation-associated keratins in benign mouse skin tumors. Carcinogenesis (Lond.), 12: 2063-2067, 1991.[Abstract/Free Full Text]
  47. Dlugosz A. A., Cheng C., Williams E. K., Dharia A. G., Denning M. F., Yuspa S. H. Alterations in murine keratinocyte differentiation induced by activated rasHa genes are mediated by protein kinase C-{alpha}. Cancer Res., 54: 6413-6420, 1994.[Abstract/Free Full Text]
  48. Leung D. W., Cachianes G., Kuang W. J., Goeddel D. V., Feder J., Connolly D. T. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science (Washington DC), 246: 1306-1309, 1989.[Abstract/Free Full Text]
  49. Keck P. J., Hauser S. D., Krivi G., Sanzo K., Warren T., Feder J., Connolly D. T. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science (Washington DC), 246: 1309-1312, 1989.[Abstract/Free Full Text]
  50. Senger D. R., Brown L. F., Claffey K. P., Dvorak H. F. Vascular permeability factor, tumor angiogenesis and stroma generation. Invasion Metastasis, 14: 385-394, 1994.[Medline]
  51. Berse B., Brown L. F., Van de Water L., Dvorak H. F., Senger D. R. Vascular permeability factor (vascular endothelial growth factor) gene is expressed differentially in normal tissues, macrophages, and tumors. Mol. Biol. Cell, 3: 211-220, 1992.[Abstract]
  52. Plate K. H., Breier G., Weich H. A., Risau W. Vascular endothelial growth factor is a potential tumor angiogenesis factor in human gliomas in vivo.. Nature (Lond.), 359: 845-848, 1992.[Medline]
  53. Shima D. T., Kuroki M., Deutsch U., Ng Y-S., Adamis A. P., D’Amore P. A. The mouse gene for vascular endothelial growth factor. J. Biol. Chem., 271: 3877-3883, 1996.[Abstract/Free Full Text]
  54. Larcher F., Robles A. I., Duran H., Murillas R., Quintanilla M., Cano A., Conti C. J., Jorcano J. L. Up-regulation of vascular endothelial growth factor/vascular permeability factor in mouse skin carcinogenesis correlates with malignant progression state and activated H-ras expression levels. Cancer Res., 56: 5391-5396, 1996.[Abstract/Free Full Text]
  55. Bizub D., Wood A. W., Skalka A. M. Mutagenesis of the Ha-ras oncogene in mouse skin tumors induced by polycyclic aromatic hydrocarbons. Proc. Natl. Acad. Sci. USA, 83: 6048-6052, 1986.[Abstract/Free Full Text]
  56. Roop D. R., Lowy D. R., Tambourin P. E., Strickland J., Harper J. R., Balaschak M., Spangler E. F., Yuspa S. H. An activated Harvey ras oncogene produces benign tumours on mouse epidermal tissue. Nature (Lond.), 323: 822-824, 1986.[Medline]
  57. Fuller J. M., Gerner E. W., Russell D. H. Polyamine biosynthesis and accumulation during the G1 to S Phase transition. J. Cell Physiol., 93: 81-87, 1977.[Medline]
  58. Kaczmarek L., Calabretta B., Ferrari S., Riel J. K. Cell cycle dependent expression of human ornithine decarboxylase. J. Cell Physiol., 132: 545-551, 1987.[Medline]
  59. Gilmour S. K., Birchler M., Smith M. K., Rayca K., Mostochuk J. Effect of elevated levels of ornithine decarboxylase on cell cycle progression in skin. Cell Growth Differ., 10: 739-748, 1999.[Abstract/Free Full Text]
  60. Megosh L., Halpern M., Farkash E., O’Brien T. G. Analysis of ras gene mutational spectra in epidermal papillomas from K6/ODC transgenic mice. Mol. Carcinog., 22: 145-149, 1998.[Medline]
  61. Klein-Szanto A. J. P. Pathology of human and experimental skin tumors Conti C. eds. . Skin Tumors: Experimental and Clinical Aspects, : 19-53, Raven Press New York 1989.
  62. Vojtek A. B., Der C. J. Increasing complexity of the Ras signaling pathway. J. Biol. Chem., 273: 19925-19928, 1998.[Free Full Text]
  63. Khwaja A., Rodriquez-Viciana P., Wennström S., Warne P. H., Downward J. Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway. EMBO J., 16: 2783-2793, 1997.[Medline]
  64. O’Reilly M. S., Holmgren L., Chen C. C., Folkman J. Angiostatin induces and sustains dormancy of human primary tumors in mice. Nat. Med., 2: 689-692, 1996.[Medline]
  65. O’Reilly M. S., Boehm T., Shing Y., Fukai N., Vasios G., Lane W. S., Flynn E., Birkhead J. R., Olsen B. R., Folkman J. Endostatin. An endogenous inhibitor of angiogenesis and tumor growth. Cell, 88: 277-285, 1997.[Medline]
  66. Folkman J. Tumor angiogenesis. Adv. Cancer Res., 48: 175-203, 1985.
  67. Ausprunk D. H., Folkman J. Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc. Res., 14: 53-65, 1977.[Medline]
  68. Risau W. Angiogenic growth factors. Prog. Growth Factor Res., 2: 71-79, 1990.[Medline]
  69. Takigawa M., Enomoto M., Nishida Y., Pan H-O., Kinoshita A., Suzuki F. Tumor angiogenesis and polyamines: {alpha}-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, inhibits B16 melanoma-induced angiogenesis in ovo and the proliferation of vascular endothelial cells in vitro. Cancer Res., 50: 4131-4138, 1990.[Abstract/Free Full Text]
  70. Sunkara P., Rosenberger A. Antimetastatic activity of DL-{alpha}-difluoromethylornithine, an inhibitor of polyamine biosynthesis, in mice. Cancer Res., 47: 933-935, 1987.[Abstract/Free Full Text]
  71. Auvinen M., Laine A., Paasinen-Sohns A., Kangas A., Kangas L., Saksela O., Andersson L. C., Hölttä E. Human ornithine decarboxylase-overproducing NIH3T3 cells induce rapidly growing, highly vascularized tumors in nude mice. Cancer Res., 57: 3016-3025, 1997.[Abstract/Free Full Text]
  72. Clifford A., Morgan D., Yuspa S. H., Peralta Soler A., Gilmour S. K. Role of ornithine decarboxylase in epidermal tumorigenesis. Cancer Res., 55: 1680-1686, 1995.[Abstract/Free Full Text]
  73. Kandel J., Bossy-Wetzel E., Radvany F., Klagsburn M., Folkman J., Hanahan D. Neovascularization is associated with a switch to the export of bFGF in the multistep development of fibrosarcoma. Cell, 66: 1095-1104, 1991.[Medline]
  74. Good D. J., Polverini P. J., Rastinejad F., LeBeau M. M., Lemons R. S., Frazier W. A., Bouck N. P. A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally distinguishable from a fragment of thrombospondin. Proc. Natl. Acad. Sci. USA, 87: 6624-6628, 1990.[Abstract/Free Full Text]
  75. Carmellet P., Ferreira V., Breier G., Pollefeyt S., Kiecksens L., Gersenstein M., Fahrig M., Vandenhoeck A., Harpal K., Eberhardt C., Declercq C., Pawling J., Moons L., Collen D., Risau W., Nagy A. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature (Lond.), 380: 435-439, 1996.[Medline]
  76. Ferrara N., Carver-Moore K., Chen K., Dowd M., Lu L., O’Shea K. S., Powell-Braxton L., Hillan K. J., Moore M. W. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature (Lond.), 380: 389-442, 1996.
  77. Shi Y. P., Ferrara N. Oncogenic ras fails to restore an in vivo tumorigenic phenotype in embryonic stem cells lacking vascular endothelial growth factor (VEGF). Biochem. Biophys. Res. Commun., 254: 480-483, 1999.[Medline]
  78. Kyriakides T. R., Tam J. W. Y., Bornstein P. Accelerated wound healing in mice with a disruption of the thrombospondin 2 gene. J. Investig. Dermatol., 113: 782-787, 1999.[Medline]
  79. Fukumura D., Xavier R., Sugiura T., Chen Y., Park E-C., Lu N., Selig M., Nielsen G., Taksir T., Jain R. K., Seed B. Tumor induction of VEGF promoter activity in stromal cells. Cell, 94: 715-725, 1998.[Medline]
  80. Olumi A. F., Grossfeld G. D., Hayward S. W., Carroll P. R., Tisty T. D., Cunha G. R. Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium. Cancer Res., 59: 5002-5011, 1999.[Abstract/Free Full Text]



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