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Carcinogenesis |
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.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-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 |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. 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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. Tumors >1 cm2 almost completely regressed
within 2–4 weeks of DFMO treatment, with no apparent toxicity to the
animals. Table 1
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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. 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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. 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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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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. Immunoblot analyses revealed that keratin 1 expression is
elevated in the epidermis of K6/ODC transgenic mice (Fig. 4b)
. 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)
. 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)
.
Interestingly, inhibiting ODC enzyme activity via treatment with DFMO
resulted in less keratin 1 expression in the regressed tumors (Fig. 4)
.
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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; Table 3
).
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)
. Table 4
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)
.
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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. 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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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Grant CA70739 from the
National Cancer Institute. 
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
3 The abbreviations used are: ODC, ornithine
decarboxylase; DFMO, -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.
Received 3/23/00. Accepted 8/25/00.
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H. Rebel, H. van Steeg, R. B. Beems, R. Schouten, F. R. de Gruijl, and C. Terleth Suppression of UV Carcinogenesis by Difluoromethylornithine in Nucleotide Excision Repair-deficient Xpa Knockout Mice Cancer Res., March 1, 2002; 62(5): 1338 - 1342. [Abstract] [Full Text] [PDF]
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C. A. Hobbs, B. A. Paul, and S. K. Gilmour Deregulation of Polyamine Biosynthesis Alters Intrinsic Histone Acetyltransferase and Deacetylase Activities in Murine Skin and Tumors Cancer Res., January 1, 2002; 62(1): 67 - 74. [Abstract] [Full Text] [PDF]
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D. J. Feith, L. M. Shantz, and A. E. Pegg Targeted Antizyme Expression in the Skin of Transgenic Mice Reduces Tumor Promoter Induction of Ornithine Decarboxylase and Decreases Sensitivity to Chemical Carcinogenesis Cancer Res., August 1, 2001; 61(16): 6073 - 6081. [Abstract] [Full Text] [PDF]
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