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[Cancer Research 62, 741-746, February 1, 2002]
© 2002 American Association for Cancer Research

Experimental Therapeutics

1{alpha},25-(OH)2-D3 and Its Synthetic Analogue Decrease Tumor Load in the Apcmin Mouse1

Sergio Huerta, Ronald W. Irwin, David Heber, Vay Liang W. Go, H. Phillip Koeffler, Milan R. Uskokovic and Diane M. Harris2

University of California-Los Angeles Center for Human Nutrition, Los Angeles, California 90095 [S. H., R. W. I., D. H., V. L. W. G., D. M. H.]; Division of Hematology-Oncology, Cedars-Sinai Medical Center/University of California-Los Angeles School of Medicine, Los Angeles, California 90048 [H. P. K.]; Hoffmann-LaRoche Inc., Nutley, New Jersey 07110 [M. R. U.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Both calcium and vitamin D are thought to be able to inhibit colon carcinogenesis. To better define the effects of vitamin D, we studied 1{alpha},25-(OH)2-D3 and a noncalcemic synthetic analogue of vitamin D3 (VD3) in the Apcmin mouse. Female Apcmin mice 4–5 weeks old were randomized to four groups: a VD3-treated group (n = 11) were given injections of 0.01 µg of 1{alpha},25-(OH)2-D3 i.p. three times per week; an analogue-treated group (n = 10) received 5 µg of 1{alpha},25-(OH)2-16-ene-19-nor-24-oxo-D3 i.p. three times per week; and a control group (n = 12) received sham injections of PBS. A sulindac-treated group (n = 10) was used as a positive control. Doses of these compounds were chosen based on previous toxicity studies in mice and rats. After 10 weeks of treatment, mice were killed and two observers (S. H., R. W. I.), blinded to treatment, scored polyp number and size. Tumor number was not affected with 1{alpha},25-(OH)2-D3 or vitamin D analogue administration. A significant decrease in total tumor load (sum of all polyp areas) over the entire gastrointestinal tract was seen in the analogue (36% decrease; P < 0.05) and the VD3 groups (46%; P < 0.001). There was a significant decrease in polyp number (49%; P < 0.001) and polyp area (70%; P < 0.001) in the sulindac group. Reverse transcription-PCR of the total RNA derived from intestinal tissue revealed expression of the vitamin D receptor throughout the small intestine and the colon. Serum calcium levels in the analogue group were not elevated at week 4 of treatment and only moderately elevated (22%) by week 8 (P <= 0.001). In contrast, serum calcium in the VD3 group was significantly elevated (P <= 0.001) at weeks 4 (23%) and 8 (45%). Food intake and growth rate were significantly lower in the VD3 group (26%, P < 0.001, and 27%, P < 0.001, respectively) at week 10. In contrast, food intake and growth rate were similar for the control, sulindac, and analogue groups. Our results indicate that a noncalcemic analogue of vitamin D can significantly decrease intestinal tumor load in Apcmin mice without severe toxic side effects and suggest that these compounds may have utility as chemopreventive agents in groups at high-risk for colon cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Last year, more than 56,000 Americans died from colorectal cancer (1) , making it the second leading cause of cancer death in men and women in the United States. About 20% of colorectal cancer cases have a familial pattern; the rest are assumed to be sporadic (2) . Human epidemiological studies provide evidence for an inverse correlation between vitamin D levels and the risk of colon cancer. One of the first observations to suggest such a relationship is that areas in the United States with the highest mean solar radiation have the lowest age-adjusted rates of colon cancer for white men (3) . Other cohort and case-control studies have been conducted to assess the role of calcium and VD33 in colon cancer prevention (4, 5, 6, 7, 8, 9) . Two of these cohort studies (10) and one case-control study (11) demonstrate a relationship between vitamin D intake and colon cancer risk. Recent studies show that decreased plasma levels of 1{alpha},25-(OH)2-D3 and 25-(OH)-D3 are associated with an increased incidence of polyp formation in the distal colon in women (12) . In contrast, diets deficient in vitamin D increase hyperplasia and hyperproliferation of colonic crypts (13) .

Vitamin D is an important hormone playing a crucial role in the maintenance of calcium homeostasis. Vitamin D is either made from cholesterol precursors in the skin or consumed in the diet from plant sources or fortified foods. Whether vitamin D is from endogenous or exogenous sources, it is hydroxylated at carbon 25 by the liver and at carbon 1 by the kidney to generate its most potent form, 1{alpha},25-(OH)2-D3. The primary role of vitamin D is to facilitate absorption of calcium and phosphate in the intestine. However, the VDR is present in many tissues and 1{alpha},25-(OH)2-D3 and several of its analogues have antiproliferative properties against many cancers in vitro, including colon (14, 15, 16, 17) , breast (18 , 19) , prostate (20 , 21) , and hematopoietic cells (22) . However, the hypercalcemic effects that result from the doses required to inhibit proliferation decrease the therapeutic usefulness of the native ligand in oncology.

Synthetic analogues that are similar in structure to 1{alpha},25-(OH)2-D3 retain the anticancer properties of the native ligand with minimal effects on calcium homeostasis. Compounds with alterations in rings A and D (Fig. 1A)Citation and the addition of double and triple bonds have been studied in HL-60 cells (23) . Studies using the first compounds developed show that the addition of a double bond on the 16th carbon of the 1{alpha},25-(OH)2-D3 skeleton (16-ene metabolites) enhance the potency of this compound by 100-fold compared with 1{alpha},25-(OH)2-D3 in the inhibition of the proliferation of HL-60 cells. The antiproliferative effect of this dose is accompanied by only a minimal effect on calcium homeostasis (23 , 24) . Analogues lacking carbon 19 of the 1{alpha},25-(OH)2-D3 skeleton (19-nor metabolites) bind the VDR with lower affinity than does the native ligand, yet they show similar antiproliferative properties. The inhibitory effect of these analogues on proliferation in the HL-60 cells is caused by a block in the transition of cells from the G0-G1 phase (25) . In addition, the removal of carbon 19 of the native ligand results in a compound with antiproliferative effects against prostate cancer cells resistant to other vitamin D analogues (26) . Similar studies demonstrate that the addition of a keto terminus at position 24 of the 1{alpha},25-(OH)2-D3 skeleton (24-oxo metabolites) yields compounds with antiproliferative properties equal to 1{alpha},25-(OH)2-D3 in prostate cancer and leukemia cells (20) , yet cause much less hypercalcemia than does 1{alpha},25-(OH)2-D3. The 1{alpha},25-(OH)2-16-ene-19-nor-24-oxo-D3 (Ro 26-9114) used in this study is a composite of these analogues (Fig. 1B)Citation , and has not previously been studied in an in vivo system.



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  		Fig. 1. A, structure of 1{alpha},25-(OH)2-D3. Carbons are numbered relative to the cholesterol precursor. B, structure of the vitamin D analogue 1{alpha},25-(OH)2-16-ene-19-nor-24-oxo-D3. The differences from the parent compound are highlighted: the 24-oxo and the 16-ene by the dashed lines and the 19-nor by the arrow.

 
Apcmin is a mouse model of intestinal carcinogenesis originally derived from a germ-line mutation induced in founder animals by ethylnitrosourea treatment (27) . The Min mutation creates a stop codon, resulting in a truncated protein. Homozygous Min/Min mice die as embryos, but on the C57BL/6J (or B6) background, Min/+ mice develop multiple intestinal neoplasias throughout the intestinal tract within a few weeks after birth. The mutation in this mouse model is analogous to that found in the human disease familial adenomatous polyposis coli, an inherited form of colorectal cancer. However, because a mutation of the APC gene is one of the earliest gene mutations that occurs in the adenoma-carcinoma sequence even in sporadic tumors (28, 29, 30) , observed effects of chemoprevention agents in this model may be applicable to both sporadic and inherited forms of colorectal cancer.

Vitamin D has been proposed to play a role in colon carcinogenesis and the Apcmin mouse bears a genetic alteration resulting in precancerous lesions. Therefore, we chose to use Apcmin to test the effectiveness of vitamin D and its analogues in colon cancer chemoprevention. The aim of our study was to assess the effects of the vitamin D analogue Ro 26-9114 in comparison with 1{alpha},25-(OH)2-D3 and the nonsteroidal anti-inflammatory drug sulindac on polyp formation in the Apcmin mouse.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Experimental Design.
Female C57BL/6J-Min/+ Apcmin mice from our breeding colony were selected for this study. We established our colony from founder mice obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were weaned at 4–5 weeks of age, at which time they were genotyped by PCR analysis, and Min/+ mice were randomized to one of four treatment groups. All of the animals were fed a standard AIN-93G purified diet (Dyets, Inc., Bethlehem, PA) and offered distilled water ad libitum.

The analogue-treated group (n = 10) received injections of 5 µg of 1{alpha},25-(OH)2-16-ene-19-nor-24-oxo D3 (a gift from Hoffmann-LaRoche) i.p. three times a week. The dose chosen had maximal antiproliferative properties and minimal toxicity in mice (24 , 31 , 32) and rats (17) in experiments evaluating various VD3 analogues. The VD3-treated group (n = 11) was given injections of 0.01 µg of 1{alpha},25-(OH)2-D3 (Sigma-Aldrich, St. Louis, MO) i.p. three times a week. The dose of VD3 was selected based on previous in vivo studies that assessed the antiproliferative properties and toxicity in dose-response studies in mice (24) and rats (33) . The nonsteroidal anti-inflammatory drug (NSAID) sulindac (160 ppM) has been shown to reduce polyp formation by as much as 80–90% in the Apcmin mouse (34 , 35) . For this reason, we administered sulindac (Sigma-Aldrich) to a positive control group (n = 10) as a validation of the experimental protocol. Sulindac was provided in drinking water (160 ppM) as described previously (36) . A negative control group (n = 12) received sham-injections of PBS (Life Technologies, Inc., Rockville, MD). Both vitamin D and the analogue were dissolved in absolute ethanol at 10-3 M and stored at -20°C before dilution with PBS to the working concentrations (24 , 37) . The maximal dose of ethanol that animals were exposed to (0.0001% v/v) has been shown previously to have no effect on tumor growth in rats (33) . Body weight and food intake were recorded two times a week and averaged to one value a week. Blood was collected by periorbital bleeding at 0, 4, and 8 weeks for measurement of serum calcium levels. All animal-use protocols were approved by the University of California-Los Angeles Animal Care and Use Committee and were in accordance with the NIH Guide for the Care and Use of Laboratory Animals (38) .

Gross Tumor Examination.
After 10 weeks of treatment, animals were killed by exsanguination under isoflurane (Abbot Laboratories, North Chicago, IL) anesthesia. The entire gastrointestinal tract was dissected as described previously (39) and immediately placed in PBS (Life Technologies, Inc.) that had been precooled to 4°C. For polyp scoring, each intestinal tract was opened longitudinally and carefully washed with PBS (Life Technologies, Inc.) for 30 s. The entire large intestine was mounted on bilubous paper (VWR Scientific Products, Westchester, PA) with the luminal side exposed. The small intestine was divided into three 4-cm segments: proximal (4 cm of small intestine distal to the stomach); medial (4 cm midway between the stomach and the cecum); and distal (4 cm adjacent to the cecum). The three segments of the small intestine were mounted for scoring in the same fashion as the large intestine.

Tissue segments were fixed in 10% buffered formalin (Biochemical Sciences, Inc., Swedesboro, NJ), dehydrated with 70% ethanol, and stained with 0.05% Coomassie Blue in PBS (Life Technologies, Inc.) for 10 min. Two observers blinded to treatment (S. H., R. W. I.) recorded the number and diameter of polyps in each portion of the gastrointestinal tract using a light microscope at x20 (Olympus SZX12, Melville, NY).

The area of each polyp was estimated from the diameter, assuming that the polyp was a perfect circle (Fig. 2)Citation . To validate this technique, the macroscopic appearance of a subset of tumors was compared with histopathological sections demonstrating that these tumors were flat (data not shown). The total polyp number is reported as the sum of the polyp number in each segment averaged over all of the animals. Total tumor load is reported as the average of the sums of the areas of the polyps in each animal.



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  		Fig. 2. Example polyp from the medial segment of the small intestine in an Apcmin mouse. This image was captured at x20 using a light microscope. The tissue was stained with 0.05% Coomassie Blue. The polyp shown has a maximum diameter of 1 mm.

 
Serum Calcium Determination.
Serum calcium was determined using a commercially available kit (Sigma Diagnostics Kit 587; Sigma-Aldrich) following the manufacturer’s protocol. Ten µl of serum collected at 0, 4, and 8 weeks of treatment or 10 µl of standard was mixed with 1 ml of the assay buffer provided in the kit. Absorbance was read at 575 nm 3 min after the addition of the buffer (to ensure completion of theo-cresolphthalein reaction). Sample concentrations were calculated relative to values for standards provided in the kit.

VDR Determination.
Expression of the VDR along the different segments of the small intestine and the colon was determined by RT-PCR as described previously (40) . Briefly, total RNA was extracted from proximal, medial, and distal samples of the small intestine and the entire colon with Qiagen RNeasy Total RNA Isolation kit (Qiagen Inc., Valencia, CA). Random hexamers (Life Technologies, Inc.) were used for priming first-strand cDNA. Synthesis of cDNA was performed using a Superscript Choice System (Life Technologies, Inc.). To amplify a 180-bp segment of the murine VDR, we used the following primer set: sense, 5'-CAT GGC CAT CTG CAT TGT CTC CCC AG-3'; and antisense, 5'-GCT TCG CAG GTC AGC CAG CTT CTG GA-3'. The positive control was amplified by PCR using primers specific for a mouse housekeeping gene, TCRd: sense, 5'-CAA ATG TTG CTT GTC TGG TG-3'; and antisense, 5'-GTC AGT CGA GTG CAC AGT TT-3' (product size, 200 bp). A negative control for the RT-PCR was included by running parallel reactions for each cDNA with TCRd control primers and exclusion of the reverse transcriptase enzyme. PCR conditions were: 94°C for 30 s, 52°C for 30 s, and 72°C for 30 s for 30 cycles. PCR products were separated on a 2% agarose gel containing ethidium bromide and visualized under UV light. The intensity of each band was evaluated by an image analyzer (Bio-Rad, Burlington, MA) and the intensity of the VDR band was compared with that of the control. The VDR RT-PCR product was confirmed by sequencing (DNA Sequencer Pharmacia A.L.F., Piscataway, NJ) and comparison to the GenBank entry.

Statistical Analysis.
PRISM statistical analysis software (GraphPad Software, Inc., San Diego, CA) was used for statistical analysis. Data are expressed as means ± SE. Treatment differences polyp number, tumor load, and serum calcium (at 0, 4, or 8 weeks) were analyzed by one-way ANOVA. Treatment differences in body weight and food intake were also evaluated by one-way ANOVA at week 10 of treatment. Dunnett’s multiple-comparison test was used to identify significant treatment effects (P <= 0.05).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Polyp Number.
No effects of the vitamin D compounds were found on the total number of polyps evaluated over all of the segments combined (Table 1)Citation . That is, the mean of the total number of polyps summed over all of the 4 segments evaluated in each animal was not significantly different between treatment groups. Sulindac treatment resulted in a 49% decrease in total tumor number (P <= 0.001).


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  		Table 1 Effects of treatment with VD3 and an analogue of VD3 (Ro 26-9114) on polyp number and tumor load in each of the intestinal segments of the small intestine and all the large intestine in Apcmin mice

 
Tumor Load.
Total tumor load was analyzed in each mouse by adding the estimates of individual polyp areas in each segment of the intestine (Table 1)Citation . Both of the vitamin D compounds decreased total tumor load. We found a 36% decrease in total tumor load with the administration of the analogue (P <= 0.05) and a 46% decrease with VD3 administration (P <= 0.001). Sulindac treatment resulted in a 70% decrease in tumor load (P <= 0.001) over the entire intestine.

Food Intake and Body Weight.
Food intake was similar in the control and the sulindac-treated groups (Fig. 3)Citation and was only slightly lower than control values in the analogue-treated group at week 10 (P <= 0.05). However, this small decrease in food intake did not affect body weight gain (Fig. 4)Citation . In contrast, a marked decrease in food intake was seen in the VD3-treated group by the end of the treatment period (Fig. 3)Citation . This effect was accompanied by a significantly lower body weight in the VD3-treated group by week 10 of treatment (Fig. 4)Citation .



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  		Fig. 3. Effect of treatment with 1{alpha},25-(OH)2-D3, its analogue, and sulindac on food intake in Apcmin mice. Food intake was recorded twice a week and averaged for the week. The four groups depicted are: control ({blacksquare}), sulindac-treated ({bullet}), analogue-treated ({blacktriangleup}), and VD3-treated ({blacktriangledown}). Treatment with vitamin D compounds resulted in 6% (analogue: *, P < 0.05) and 15% (VD3: **, P < 0.001) decrease in food intake at week 10.

 


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  		Fig. 4. Effect of treatment with 1{alpha},25-(OH)2-D3 and its analogue on body weight in Apcmin mice. Body weight was recorded twice a week and averaged for the week. The four groups depicted are: control ({blacksquare}), sulindac-treated ({bullet}), analogue-treated ({blacktriangleup}), and VD3-treated ({blacktriangledown}). 1{alpha},25-(OH)2-D3 treatment resulted in a 24% decrease in body weight compared with the control group (**, P < 0.001).

 
Serum Calcium.
Serum calcium levels in the group receiving the vitamin D analogue were not altered at week 4 of treatment compared with the control group, and were only moderately elevated (22%) by the 8th week of treatment (P <= 0.001). In contrast, there was a significant 23% and 45% increase relative to control in serum calcium in the VD3-treated group starting at weeks 4 and 8, respectively (P <= 0.001; Fig. 5Citation ). Sulindac treatment had no effect on serum calcium levels compared with control.



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  		Fig. 5. Effect of treatment on serum calcium levels measured at weeks 0, 4, and 8. Analogue treatment resulted in a 22% increase at week 8 (**, P < 0.001). 1{alpha},25-(OH)2-D3 treatment resulted in 23% increase at week 4 and a 45% at week 8 (**, P < 0.001).

 
VDR Expression.
RT-PCR analysis revealed VDR expression in all segments of the small intestine and in the colon (Fig. 6)Citation . Densitometry analysis of the gel image revealed no difference in the intensity of the control versus VDR bands in any of the segments of the small intestine or the colon.



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  		Fig. 6. VDR expression in various segments of the small intestine and the colon. All of the segments of the small intestine reveal expression of the VDR as shown by the presence of the 180-bp product. Presence of product amplified from the control primers (TCRd) was observed in all segments of the small intestine and the colon.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Despite the reasonably consistent evidence from studies in vitro that vitamin D mitigates proliferation and induces terminal differentiation of normal, colon adenoma, and cancer cells, there is only limited evidence of a protective effect of vitamin D on colorectal adenoma and colorectal cancer in human studies. For instance, vitamin D and its metabolites ameliorated hyperplasia in patients with ulcerative colitis (41) , and 1{alpha},25-(OH)2-D3 reduced crypt cell production in colonic tissue removed from individuals with familial adenomatous polyposis (42) . Experiments in several animal models of cancer also show the ability of vitamin D to prevent or treat tumors. For example, i.p. administration of 1{alpha},25-(OH)2-D3 suppressed growth of human colon tumor xenografts in nude mice (43) . Administration of vitamin D prior to a carcinogenic insult significantly reduced the incidence of colonic tumors in rats (33) . Mouse studies also demonstrated that vitamin D supplementation diminished intestinal cellular proliferation induced by fatty acids (44) , cholic acid (45 , 46) , or a "Western diet" (high fat and phosphate and low calcium and vitamin D; Refs. 47 , 48 ). Duration of survival was also prolonged in leukemic animals treated with 1{alpha},25-(OH)2-D3 (32) .

The present study demonstrated that the administration of 1{alpha},25-(OH)2-D3 and its analogue resulted in a significant decrease in total intestinal tumor load in Apcmin mice. These findings are consistent with the in vitro antiproliferative properties of VD3 and VD3 analogues observed in colon cancer cells (14, 15, 16) . Although administration of both 1{alpha},25-(OH)2-D3 and the analogue that we tested resulted in decreased tumor load, we observed mild negative side effects with the administration of the analogue, and the native ligand resulted in decreased food intake and body weight. The observed decrease in caloric intake in the VD3-treated animals could be partially responsible for the decrease in tumor load in this group. However, because treatment with the analogue also produced a similar decrease in tumor load, we think that both of the vitamin D compounds have antiproliferative properties. In this mouse model with a germ-line mutation, the reduction in tumor load rather than in tumor number suggests that 1,25 (OH)2-D3 and its synthetic analogue prevent tumor progression, rather than adenoma initiation.

The antiproliferative properties of 1{alpha},25-(OH)2-D3 in the colonocyte may be mediated by effects on calcium homeostasis, by alterations in gene expression regulated through the intracellular VDR (21) , or by mechanisms yet to be elucidated. Studies in vitro demonstrated that the antiproliferative effects of 1{alpha},25-(OH)2-D3 occurred only in colon cancer cell lines that expressed the VDR (15 , 21) . In the present study, we demonstrated that the VDR is uniformly distributed in all segments of the small intestine and the colon, which was consistent with observations in other reports that described an equal distribution of the VDR protein in ascending and descending colon in mice (49) . Despite equal VDR distribution, there was a different response to treatment with the vitamin D compounds along the various segments of the small intestine and the colon (Table 1)Citation . It is unclear why such differences were observed. However, topical distribution of human colon cancers is variable, and the mechanism of this phenomenon is also unclear. An analysis of proliferative and apoptotic indices along the various intestinal segments in our mouse model is under way.

The toxicity observed with 1{alpha},25-(OH)2-D3 paralleled the degree of hypercalcemia observed in these animals. The observed hypercalcemia in our analysis is consistent with findings of other studies and prohibits the use of the native ligand as a therapeutic agent in the prevention of colon carcinogenesis. For instance, individuals with preleukemia who were given 1{alpha},25-(OH)2-D3 developed serum hypercalcemia at concentrations that were lower than the optimal concentrations determined to maximally inhibit proliferation of hematopoietic cells in vitro (50) . This toxicity has driven the development of several vitamin D analogues with maximal antiproliferative properties and minimal hypercalcemic effects (20 , 23 , 24) .

Administration of the VD3 analogue used in this study resulted in a diminished hypercalcemic effect when compared with the native ligand, and this effect began later in the treatment period (8th week). The moderate degree of hypercalcemia in the VD3 analogue-treated group was not accompanied by a significant effect on weight gain and only a minimal effect on food intake. This short-term study did not address the potential toxicity of this analogue with its chronic use. However, our results indicate that the antitumor properties of Ro 26-9114 are retained with decreased toxic side effects compared with the native ligand. Studies to assess the optimal dose of Ro 26-9114 and to determine the mechanism of action of this analogue are continuing.

In this study of the Apcmin mouse model of FAP, we observed a significant reduction in tumor load with the administration of 1{alpha},25-(OH)2-D3 and its synthetic analogue. These findings suggest that vitamin D compounds have antitumor properties even in preexisting adenomas and that it is possible to retain the antiproliferative properties of 1{alpha},25-(OH)2-D3 while minimizing the hypercalcemic toxicity by use of a synthetic analogue. The use of this analogue is promising as a preventive agent in patients with familial adenomatous polyposis or in similar high-risk groups.


   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 by the Cancer Research Fund, under Interagency Agreement 97-12013 (University of California contact 98-00924V) with the Department of Health Services, Cancer Research Program. Additional funding and support was obtained from the University of California-Los Angeles Clinical Nutrition Research Unit (NIH Grant CA42710). Back

2 To whom requests for reprints should be addressed, at UCLA Center for Human Nutrition, 900 Veteran Avenue, 13-145 Warren Hall, Box 95142, Los Angeles, California 90095-1742. Phone: (310) 206-7281; Fax: (310) 824-5990; E-mail: dmharris{at}ucla.edu Back

3 The abbreviations used are: VD3, vitamin D3; VDR, vitamin D receptor; RT-PCR, reverse transcription-PCR; TCRd, T-cell receptor, delta chain. Back

Received 8/21/01. Accepted 12/ 3/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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