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Molecular Cancer Research 1:56-67 (2002)
© 2002 American Association for Cancer Research

Signaling and Regulation

Targeted Disruption of the 25-Hydroxyvitamin D3 1{alpha}-Hydroxylase Gene in ras-Transformed Keratinocytes Demonstrates That Locally Produced 1{alpha},25-Dihydroxyvitamin D3 Suppresses Growth and Induces Differentiation in an Autocrine Fashion1

Dao Chao Huang1, Vasilios Papavasiliou1, Johng S. Rhim2, Ronald L. Horst3 and Richard Kremer1

1 Department of Medicine, Royal Victoria Hospital and McGill University, Montreal, Quebec, Canada;
2 Center for Prostate Disease Research, Uniformed Services, University of the Health Sciences, Bethesda, MD; and
3 USDA, Agriculture Research Service, National Animal Disease Center, Ames, IA

Requests for reprints: Richard Kremer, Room H4.67, Calcium Research Laboratory, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, H3A 1A1 Canada. Phone: (514) 843-1632; Fax: (514) 843-1712.


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
It has been previously shown that keratinocytes express a high level of 25-hydroxyvitamin D3 (25-OHD3) 1{alpha}-hydroxylase (1{alpha}-hydroxylase). 1{alpha}-Hydroxylase catalyzes the conversion of 25-OHD3 to 1{alpha},25-dihydroxyvitamin D3 [1,25(OH)2D3]. 1,25(OH)2D3 is both antiproliferative (i.e., suppresses cell growth) and prodifferentiative (i.e., induces cell differentiation) in many cell types. We hypothesized that local production of 1,25(OH)2D3 by keratinocytes may suppress their growth and induce their differentiation in an autocrine fashion. To test this hypothesis, we inactivated both 1{alpha}-hydroxylase alleles in a ras-transformed keratinocyte cell line, HPK1Aras, which typically produces squamous carcinoma in nude mice. To inactivate 1{alpha}-hydroxylase expression by HPK1Aras cells, we disrupted both alleles of the 1{alpha}-hydroxylase gene by homologous recombination. Lack of expression and activity of 1{alpha}-hydroxylase was confirmed by Northern blot analysis and detected conversion of 25-OHD3 to 1,25(OH)2D3. We then examined the effect of substrate 25-OHD3 on parameters of growth and differentiation in the double knockout cell line as compared to wild-type HPK1Aras cells in vitro. It was found that 1{alpha}-hydroxylase inactivation blocked the antiproliferative and prodifferentiative effect of 25-OHD3. These in vitro effects were further analyzed in vivo by injecting knockout or control cells subcutaneously in severely compromised immunodeficient mice. Tumor growth was accelerated and differentiation was inhibited in mice given injections of knockout cells as compared to control cells in the presence of substrate 25-OHD3. Our results demonstrate, for the first time, that 1{alpha}-hydroxylase expression by keratinocytes plays an important role in autocrine growth and differentiation of these cells, and suggest that expression of this enzyme may modulate tumor growth in squamous carcinomas.

Key Words: cancer • vitamin D • knockout • keratinocytes • 1{alpha}-hydroxylase


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Synthesis of 1{alpha},25-dihydroxyvitamin D3 [1,25(OH)2D3] from its endogenous precursor, 25-hydroxyvitamin D3 (25-OHD3), is catalyzed by 25-hydroxyvitamin D3-1{alpha}-hydroxylase (1{alpha}-hydroxylase), a mitochondrial cytochrome P-450 enzyme (1–3). Although the kidney is the principal site of 1,25(OH)2D3 synthesis (4–8), extrarenal production can contribute to significant amounts of circulating 1,25(OH)2D3 levels when kidney function is absent (9, 10).

Recent experiments have identified human keratinocytes as an interesting alternate source for 1,25(OH)2D3 production. In addition to producing previtamin D3 from 7-dehydrocholesterol, keratinocytes are also able to produce the biologically active metabolite, 1,25(OH)2D3 (11). Furthermore, the skin, the main source of human keratinocytes, is well established as a target tissue for 1,25(OH)2D3, because it expresses the vitamin D receptor (VDR) (12). 1,25(OH)2D3 is a strong antiproliferative agent and induces cell differentiation through its interaction with the VDR (4, 11, 13–16). In addition, 1,25(OH)2D3 has been shown to suppress tumor growth, inhibit metastasis, and prolong survival in animal models (17–19). However, the clinical use of 1,25(OH)2D3 is limited by its strong effects on calcium metabolism because a few µg/day of this compound leads to hypercalcemia (20).

Recently, 1{alpha}-hydroxylase cDNA was cloned (21, 22) and found to be expressed at high levels in keratinocytes relative to other tissues (22), suggesting that 1{alpha}-hydroxylase may play an important local role in this tissue. In the present study, we examined the growth and differentiation both in vitro and in vivo of ras-transformed keratinocytes (23) following inactivation of the 1{alpha}-hydroxylase gene. We found that these cells can effectively utilize 25-OHD3, the inactive precursor of 1,25(OH)2D3, to regulate their growth and differentiation via the 1{alpha}-hydroxylase enzyme system. Our study suggests that the expression of the 1{alpha}-hydroxylase enzyme is involved in the autocrine regulation of growth and differentiation of keratinocytes.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
1{alpha}-Hydroxylase Expression in Wild Type and Knockout Generated Cell Lines
To achieve targeted disruption of the 1{alpha}-hydroxylase gene in HPK1Aras cells, we used a homologous recombination method with isogenic DNA. The targeting constructs were generated from a 4-kb fragment of the P450c 1{alpha}-hydroxylase gene which was isolated by PCR from HPK1Aras cells. A neomycin resistance cassette (2.2 kb) flanked by BGH pA and SV40 pA was inserted between exons 2 and 3 of the 1{alpha}-hydroxylase gene using SmaI and NotI sites causing its disruption (Fig. 1, A and B ). Northern blot analysis of HPK1Aras cells revealed a 2.4-kb mRNA 1{alpha}-hydroxylase transcript (21, 22) in wild-type (WT) HPK1Aras cells (Fig. 1C, upper panel). In contrast, mRNA levels were significantly reduced in single knockout (SKO) and totally absent in double knockout (DKO) HPK1Aras cells (Fig. 1C, upper panel). Northern blot analysis also revealed a similar 2.4-kb transcript in normal human renal proximal tubule epithelial cells, in normal human epidermal keratinocytes, and in immortalized human keratinocytes (HPK1A), but not in COS-7 and PC12 cells (Fig. 1C, upper panel).




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  		FIGURE 1. Gene targeting of the human 1{alpha}-hydroxylase gene. A. Schematic representation of the gene region encoding the 1{alpha}-hydroxylase and creation of a mutant allele by homologous recombination. The neomycin resistance cassette (2.2 kb) is flanked by ~1.4 and 2.5 kb of 5' and 3' genomic sequences. B. Southern blot of genomic DNA isolated from HPK1Aras parental WT, SKO, and DKO cells. Purified DNA was digested with BamHI and hybridized with the 340-bp NotI/NcoI probe. In parental cells, a 4.2-kb BamHI fragment is detected. In the correctly targeted gene, an additional 2.2-kb fragment of neo cassette is present so that a 6.4-kb fragment is detected. C. Northern analysis of WT, SKO, and DKO HPK1Aras cell lines; human renal proximal tubule epithelial cells (R); normal human epidermal keratinocytes (K); HPK1A (1A); PC12 (C1); and COS-7 cells (C2). 1{alpha}-Hydroxylase mRNAs of 2.4 kb and VDR mRNAs of 4.6 kb are observed in human renal proximal tubule epithelial cells (R), normal human epidermal keratinocytes (K), HPK1A (1A), and WT and SKO HPK1Aras cell lines but not in PC12 and COS-7. HPK1Aras DKO cells express VDR mRNA but not 1{alpha}-hydroxylase mRNA. Cyclophilin hybridization is shown as a measure of endogenous RNA in each sample.

 
Expression of VDR and Effects of 1,25(OH)2D3 on Cell Growth in WT and KO Cells
Northern blot analysis using a VDR cDNA probe revealed similar levels of expression in SKO, DKO, and WT HPK1Aras cells (Fig. 1C, middle panel). The addition of 1,25(OH)2D3 to the culture medium produced a sustained inhibition of cell growth over time in both WT and DKO HPK1Aras cells to a similar degree (Fig. 2 ), indicating the integrity of the VDR-mediated growth inhibitor following 1{alpha}-hydroxylase gene disruption.



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  		FIGURE 2. Effect of 1,25(OH)2D3 on in vitro cell growth kinetics in WT (control) and DKO HPK1Aras cells. Cells were grown as described in "Materials and Methods." Following a 24-h incubation in serum-free conditions, fresh DMEM was added containing 10% charcoal-stripped fetal bovine serum (FBS) in the presence or absence of 0.1 µM 1,25(OH)2D3. Cell number at time 0 was 3.80 ± 0.30 x 104. Points, means of triplicate determinations; bars, SE. The graph shown is representative of three different experiments. *, significantly different from untreated cells; P < 0.005.

 
In Vitro Production of 1,25(OH)2D3 in WT, SKO, and DKO HPK1Aras Cells
To determine 1{alpha}-hydroxylase activity in these cell lines, substrate 25-OHD3 was added to the culture medium and 1,25(OH)2D3 production in the conditioned medium was examined. WT HPK1Aras converted 25-OHD3 to 1,25(OH)2D3, whereas DKO HPK1Aras cells did not (Fig. 3, A and C ). SKO HPK1Aras cells produced intermediate levels of 1,25(OH)2D3 (Fig. 3B). The identity of 1,25(OH)2D3 metabolite production was verified by high-pressure liquid chromatography (HPLC) and indicated that the product coeluted with authentic 1,25(OH)2D3 and not with any other vitamin D metabolite (Fig. 3D).



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  		FIGURE 3. In vitro production of 1,25(OH)2D3 in WT (A), SKO (B), and DKO HPK1Aras cells (C). Points, means; bars, SE. The graphs shown are representative of three different experiments. D. The fractions containing 1,25(OH)2D3 coeluting with reference crystalline 1,25(OH)2D3 in A were pooled, evaporated, and rechromatographed by HPLC. The stippled bars are 1-min fractions collected and assayed for 1,25(OH)2D3 activity as described in "Materials and Methods."

 
In Vitro Effects of 25-OHD3 on Cell Growth and the Cell Cycle
Addition of 25-OHD3 to the culture medium produced a significant inhibition of cell growth in WT HPK1Aras cells as determined by cell count (Fig. 4A ) and formazan production (Fig. 4C). In contrast, DKO HPK1Aras cells were not significantly growth inhibited by addition of substrate 25-OHD3 (Fig. 4, B and D), indicating that the expression of 1{alpha}-hydroxylase is necessary to observe this growth-inhibitory effect.



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  		FIGURE 4. Effect of 25-OHD3 on in vitro growth kinetics in WT HPK1Aras transfected with control vector pcDNA3 cells and DKO HPK1Aras cells. After 24 h in basal conditions, cells were treated with DMEM containing 10% charcoal-stripped FBS in the absence or presence of 1 µM 25-OHD3. Comparisons of cell numbers (A and B) and MTS/formazan production (C and D) show that addition of substrate 25-OHD3 significantly inhibits cell growth in WT HPK1Aras cells (A and C), but has no effect in DKO cells (B and D). Points, means of sextuplicate determinations; bars, SE. The graphs shown are representative of three different experiments. *, significantly different from untreated cells; P < 0.01.

 
In addition to its effects on cell growth, we also analyzed 25-OHD3 effects on the cell cycle progression of WT and DKO HPK1Aras cells by flow cytometry 9Fig. 5 ). Addition of 1 µM 25-OHD3 to the culture medium for 24 h resulted in a significant reduction of cells in S phase and accumulation in G0-G1 phase in WT HPK1Aras cells (Fig. 5C). In contrast, no effect was observed in DKO cells following addition of substrate 25-OHD3 (Fig. 5D). Altogether, these results suggest that local production of 1,25(OH)2D3 inhibits growth by an autocrine mechanism.



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  		FIGURE 5. Cell cycle analysis of WT (A and C) and DKO HPK1Aras cells (B and D) in the absence and presence of 1 µM 25-OHD3. After 24 h in serum-free conditions, the medium was changed and fresh DMEM supplemented with 10% charcoal-stripped FBS alone (A and B) or containing 1 µM 25-OHD3 was added (C and D), and incubation continued for 24 h. Cells were stained and analyzed by flow cytometry as described in "Materials and Methods." Results are expressed as percentage of cells distributed in G0-G1 (R1), G2-M (R2), and S phase (R3) of the cell cycle phase. Points, means of triplicate determinations. The graphs shown are representative of two different experiments.

 
In Vitro Effect of 25-OHD3 on Cell Differentiation
Both WT and DKO HPK1Aras cells treated with 1,25(OH)2D3 demonstrated strong immunoreactivity to the anti-keratin antibody AE3 with light to dark brown reactions diffused throughout the entire cytoplasm in many cells (Fig. 6, C and D ). WT HPK1Aras cells treated with 25-OHD3 displayed strong immunoreactivity similar to cells treated with 1,25(OH)2D3 (Fig. 6E). In contrast, DKO HPK1Aras cells treated with 25-OHD3 showed little or no immunoreactivity (Fig. 6F) similar to control cells (Fig. 6B). These in vitro effects were further analyzed in vivo as described below.



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  		FIGURE 6. Effect of 25-OHD3 and 1,25(OH)2D3 on HPK1Aras cell differentiation. WT and DKO HPK1Aras cells were treated with 25-OHD3 (1 µM), 1,25(OH)2D3 (0.1 µM), or vehicle alone as described in "Materials and Methods." Treatment with 1,25(OH)2D3 induced strong keratin expression in both WT and DKO HPK1Aras cells (C and D) as compared to cells treated with vehicle alone (A and B). WT HPK1Aras cells treated with 25-OHD3 demonstrated strong keratin staining similar to 1,25(OH)2D3 (C and E). In contrast, light staining was observed in DKO HPK1Aras cells treated with 25-OHD3 (F) similar to vehicle-treated cells (B). Magnification, x100; scale bar, 50 µm.

 
In Vivo Effect of 25-OHD3 on Biochemical and Physical Indices
Control (non-tumor-bearing) animals remained normocalcemic after continuous infusion of 25-OHD3 despite a 4-fold increase in their 25-OHD3 blood levels as compared with vehicle-treated animals. These 25-OHD3-treated control animals also demonstrated no difference in weight (data not shown) and no signs of distress as compared to vehicle-treated animals (see Table 1). Tumor-bearing animals treated with 25-OHD3 displayed similar physical and biochemical indices as vehicle-treated tumor-bearing animals did (see Fig. 7 ).


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  		Table 1. Plasma Calcium and 25-OHD3 Levels in 25-OHD3-Treated and Untreated Mice

Note: 25-OHD3 (0–2000 pM/24 h) was administered continuously for the duration of the experiment. Mice were bled once a week and their plasma calcium and 25-OHD3 levels were determined as described in "Materials and Methods." Results represent the means (±SE) of five mice in each group.

 


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  		FIGURE 7. Body weight (A and B) and plasma calcium (C and D) of SCID mice bearing WT or DKO HPK1Aras cells and treated with continuous infusion of 25-OHD3 (2000 pM/24 h) or vehicle using Alzet osmotic mini-pumps. Weight and plasma calcium were determined weekly as described in "Materials and Methods." No significant difference was observed between vehicle-treated and 25-OHD3-treated groups. The results are the means of 14 starting mice in each group; bars, SE.

 
In Vivo Effect of 25-OHD3 on Tumor Cell Growth
To test the ability of tumor-produced 1,25(OH)2D3 to regulate cell growth in vivo, WT and DKO HPK1Aras cells were implanted subcutaneously into severely compromised immunodeficient (SCID) mice. First, we determined the optimal infusion of 25-OHD3 to achieve maximal circulating levels of 25-OHD3 in control non-tumor-bearing animals using constant delivery of 25-OHD3 with Alzet osmotic mini-pumps. Maximal levels of 25-OHD3 were achieved with a constant infusion of 2000 pM/24 h and blood calcium levels remained normal during the infusion (see Table 1).

Mice developed palpable tumors within 2 weeks. At 2 weeks, animals received either 2000 pM/24 h of 25-OHD3 or vehicle alone by constant infusion with Alzet osmotic mini-pumps placed immediately adjacent to the tumor site for a duration of 5.5 weeks. Animals that received implants of WT or DKO HPK1Aras cells showed early and rapid tumor growth with infusion of vehicle alone. Animals that received implants of WT HPK1Aras cells and treated with 25-OHD3 showed a significant reduction in tumor growth (Fig. 8A ), whereas tumor growth could not be inhibited by 25-OHD3 infusion (Fig. 8B) in animals that received implants of DKO HPK1Aras cells. In addition, tumor weight measured at sacrifice (at 7.5 weeks) was significantly lower in animals that received implants of WT HPK1Aras cells and treated with 25-OHD3 as compared to animals treated with vehicle alone (Fig. 8C), but no such effect was observed in animals that received implants of DKO HPK1Aras cells (Fig. 8D).



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  		FIGURE 8. In vivo tumor growth kinetics in SCID mice following s.c. injection of 3 x 106 cells in PBS mixed with matrigel (1:1). 25-OHD3 (2000 pM/24 h) was administered by constant infusion using Alzet osmotic mini-pumps. SCID mice that received implants of WT control HPK1Aras/pcDNA3 cells (A) or DKO HPK1Aras cells (B). 25-OHD3 administration significantly inhibits tumor growth in animals that received implants of WT HPK1Aras cells (A and C), but has no effect in animals that received implants of DKO HPK1Aras cells (B and D). Seven and a half weeks after tumor implantation and treatment with 25-OHD3 or vehicle, mice were killed and the weight of tumors in animals implanted with WT (C) or DKO cells (D) was measured. Data are expressed as means of 12 mice in each group. This experiment was repeated twice. *, significantly different from vehicle-treated animals at each time point; P < 0.05.

 
In Vivo Effect of 25-OHD3 on Tumor Differentiation
Histology and immunostaining of tumors in nude mice derived from HPK1Aras cells were also analyzed. Tumors derived from animals that received implants of WT HPK1Aras cells and treated with 25-OHD3 showed strong staining with the anti-keratin antibody (Fig. 9F ) as compared to control tumors derived from animals treated with vehicle alone (Fig. 9E). In contrast, tumors derived from animals that received implants of DKO HPK1Aras cells and treated with 25-OHD3 showed light staining with the anti-keratin antibody (Fig. 9H) similar to tumors treated with vehicle alone (Fig. 9G). Other differences were also noted between the two tumor types by H&E staining. Tumors derived from animals that received implants of WT HPK1Aras cells and treated with 25-OHD3 were encapsulated with fibrotic tissue and formed spheroids (cribriform structures) ranging from 200 to 400 µm in diameter, harboring 300–500 cells/capsule (Fig. 9B) as compared to tumors derived from animals that received implants of WT HPK1Aras cells and treated with vehicle alone (Fig. 9A). This phenomenon was not observed in tumors derived from animals that received implants of DKO HPK1Aras cells and treated with 25-OHD3 (Fig. 9D) or in tumors derived from animals that received implants of DKO HPK1Aras cells and treated with vehicle alone (Fig. 9C).



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  		FIGURE 9. Histology and immunostaining of tumors derived from HPK1Aras cells. H&E staining of tumors is shown in panels A–D. WT HPK1Aras tumors treated with 25-OHD3 show encapsulation and cribriform structure (B). DKO HPK1Aras tumors treated with 25-OHD3 (D) are similar to vehicle-treated tumors (C). Immunostaining with the anti-keratin antibody is strong in WT HPK1Aras tumors treated with 25-OHD3 as compared to tumor treated with vehicle (E and F). In contrast, keratin expression in DKO HPK1Aras tumors treated with 25-OHD3 cells is similar to cells treated with vehicle (G and H). H&E, cribriform structure (C), encapsulation (E), fibrosis (F), and tumor (T). Magnification, x40, scale bar, 400 µm (A–D), and x100, scale bar, 200 µm (E–H).

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
In addition to the classical target tissues of vitamin D, the bone and intestine, the skin is a target organ (12, 24). Addition of 1,25(OH)2D3 to keratinocyte cultures inhibits proliferation and stimulates differentiation in combination with extracellular calcium (25, 26). In addition to expressing the VDR, keratinocytes also express 1{alpha}-hydroxylase (11, 15). Previous studies have shown that 1{alpha}-hydroxylase originating from proximal renal tubular epithelial cells is essential for the endocrine function of 1,25(OH)2D3. We found that 1{alpha}-hydroxylase is expressed in keratinocytes similar to levels observed in renal tubular epithelial cells (Fig. 1C), which suggests that this keratinocyte 1{alpha}-hydroxylase production exerts principally, if not exclusively, an autocrine role. Our study is the first to provide information on this potential autocrine mechanism.

Our initial objective was to determine if substrate 25-OHD3, the inactive precursor of 1,25(OH)2D3, had growth-inhibitory effect in our model. Indeed, 25-OHD3 significantly suppressed growth most likely through its conversion to its active metabolite, 1,25(OH)2D3. First, we observed that 25-OHD3 had a growth-inhibitory effect and prodifferentiative effect in WT HPK1Aras cells, suggesting that its conversion to 1,25(OH)2D3 was responsible for this effect. A major obstacle to these studies is the potential that the inactive precursor, 25-OHD3, may have pharmacological effects on its own or may be converted to other active metabolites independent of the 1{alpha}-hydroxylase system. To circumvent these difficulties, we inactivated both copies of the 1{alpha}-hydroxylase gene by homologous recombination using a single targeting construct introduced in a human ras-transformed keratinocyte cell line (27). Inactivation of one allele (SKO) reduced the expression by ~50%, whereas inactivation of both alleles (DKO) completely eliminated 1{alpha}-hydroxylase expression. Similar results were observed when 1{alpha}-hydroxylase activity was quantified in vitro in the conditioned medium of these cells. Because 25-OHD3 may interact with VDR at high concentrations, it was critical to evaluate the effect of 25-OHD3 on cell growth in the DKO cells which have lost their capacity to convert 25-OHD3 to 1,25(OH)2D3. No effect on cell growth in DKO cells was observed in the presence of 25-OHD3, indicating that 1{alpha}-hydroxylase expression is required for growth inhibition by 25-OHD3 in this model. Furthermore, VDR was expressed at similar levels in both WT and DKO cell lines and both cell lines responded similarly to exogenous addition of 1,25(OH)2D3, indicating the functional capacity of the VDR in both systems.

1,25(OH)2D3 exerts its antiproliferating effects by inhibiting the progression into the S phase of the cell cycle (23), an effect clearly dependent on the presence of the VDR (28–31). Therefore, we next examined the effect of 25-OHD3, the precursor of 1,25(OH)2D3, on the cell cycle. We observed that 25-OHD3 inhibited the progression into the S phase leading to accumulation of cells in G0-G1 in WT, but not in DKO HPK1Aras cells, indicating that conversion of 25-OHD3 to 1,25(OH)2D3 by HPK1Aras cells was necessary to observe these effects. In addition to its antiproliferative effect, 1,25(OH)2D3 promotes cellular differentiation of keratinocytes (14, 25). Our study indicates that similar to the effect observed with 1,25(OH)2D3, its inactive precursor, 25-OHD3, induces keratinocyte differentiation only in the presence of an active 1{alpha}-hydroxylase enzyme.

Finally, we addressed the question of whether 1{alpha}-hydroxylase expression by WT HPK1Aras cells could regulate tumor growth and differentiation in vivo similar to the effect of exogenous administration of active vitamin D in a nude mouse model that received transplants of HPK1Aras cells (32). To address this question, we infused high concentrations of 25-OHD3 immediately adjacent to the tumor site in SCID mice that received transplants of HPK1Aras cells. Similar to the effects seen in vitro, we observed a significant inhibition of tumor growth and induction of cell differentiation in animals that received implants of WT but not DKO HPK1Aras cells. Furthermore, these effects were associated with changes in the organ microenvironment and, in particular, tumor encapsulation, an effect observed previously with other active tumor antagonists (33).

Our results indicate the capacity of locally produced 1,25(OH)2D3 to inhibit cell growth and induce differentiation in an autocrine fashion. Although high circulating levels of 25-OHD3 were achieved, no significant changes in biochemical and physical indices were observed, indicating the absence of systemic effects of circulating 25-OHD3. However, the role of 1{alpha}-hydroxylase in normal tissue homeostasis may be best assessed in a KO animal model for 1{alpha}-hydroxylase. Indeed, such a model has recently been reported and may prove extremely useful when looking at skin development (34). In addition, these in vivo studies have wider implications because significant effects on tumor development correlated with 1{alpha}-hydroxylase activity. The absence of a significant inhibition in tumor growth in vivo without addition of exogenous substrate 25-OHD3 indicates that endogenous levels of 25-OHD3 were insufficient to modulate tumor growth in vivo. A consistent but small difference in tumor volume and weight was observed between KO and WT tumors in the absence of substrate 25-OHD3. However, this trend never reached statistical significance. A number of possible explanations may explain our observation. First, animals that received transplants of either WT or KO tumors produce 1,25(OH)2D3 from the kidney. This extratumoral 1,25(OH)2D3 production may influence to some extent tumor growth and the ratio between tumoral and extratumoral production of 1,25(OH)2D3 may therefore affect the differences in tumor sizes observed. Addition of substrate 25-OHD3 will favor 1,25(OH)2D3 production by tumor cells while leaving kidney production of 1,25(OH)2D3 unchanged. This is because kidney 1{alpha}-hydroxylase activity is tightly regulated by 1,25(OH)2D3 production but not tumoral 1{alpha}-hydroxylase (35). Consequently, additional substrate will increase 1,25(OH)2D3 production by tumor cells almost linearly but not by kidney cells. To determine the "true autocrine" effect of 1,25(OH)2D3 produced by tumor cells, 1{alpha}-hydroxylase activity by the kidney would have to be selectively shut down. Alternatively, tumor development could be looked at in vitamin D-deficient animals and compared to vitamin D-replete animals that received transplants of HPK1Aras tumors. Second, we previously reported that 1,25(OH)2D3 is extensively and rapidly metabolized by HPK1Aras cells (36) and 25-OHD3 substrate may therefore be necessary to provide sufficient production of 1,25(OH)2D3 by tumor cells. Endogenous circulating 25-OHD3 levels may be insufficient to achieve sufficient intratumoral levels especially as tumor growth progresses. Conversely, variable levels of 1{alpha}-hydroxylase expression by tumor cells in vitamin D-sufficient individuals may lead to variable degrees of tumor growth. A critical element required to test this hypothesis would be the determination of 1{alpha}-hydroxylase in various types of cancer. Further investigation would warrant the expression of 1{alpha}-hydroxylase in various cancer types including squamous cancers. The significance of our findings in tumor biology may come from studies determining both 25-OHD3 levels and 1{alpha}-hydroxylase activity in human samples. The recent cloning and availability of 1{alpha}-hydroxylase should be useful to address this issue.

In summary, our study demonstrates that extrarenal production of 1,25(OH)2D3 exerts a local effect on cell growth and differentiation by utilizing its inactive precursor, 25-OHD3. This local effect differs significantly from 1{alpha}-hydroxylase production by the proximal tubular cells of the kidney. 1,25(OH)2D3 production by the kidney has a systemic effect on target organs, such as the gut and skeleton, by acting as an endocrine factor (37). In eliminating 1,25(OH)2D3 production by keratinocytes using targeted disruption of the 1{alpha}-hydroxylase gene, we demonstrated an acceleration of cell proliferation and an inhibition of differentiation. Our study, therefore, provides the first evidence that 1{alpha}-hydroxylase enzyme expressed in keratinocytes controls cell growth and differentiation in an autocrine fashion.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Cell Lines and Culture Conditions
The HPK1A cell line was established from normal human keratinocytes by stable transfection with human papillomavirus type 16 and transformed by transfection with a plasmid carrying an activated H-ras oncogene to create HPK1Aras cells (23). HPK1Aras cells form colonies in soft agar, and produce invasive squamous cell carcinomas when transplanted into nude mice (38). These cell lines were grown in DMEM (Life Technologies, Inc., Burlington, ON, Canada) supplemented with 10% FBS. Normal human renal proximal tubule epithelial cells and normal human epidermal keratinocytes (neonatal skin) were obtained from Clonetics Corp. (San Diego, CA) and were grown in renal growth medium and keratinocyte growth medium, respectively, according to the manufacturer's recommendations. PC12 and COS-7 were obtained from American Type Culture Collection (Rockville, MD) and cultured in DMEM supplemented with 10% FBS. All cells were incubated at 37°C in a humidified incubator containing 5% CO2. The culture medium was changed every 2–3 days.

Cell Counts and MTS/Formazan Assay
For MTS/formazan assay (Promega, Madison, WI), cells were seeded at a density of 5 x 103 cells/100 µl into 96-well microtiter plates and incubated for 24 h in DMEM containing 10% charcoal-stripped FBS. Following a 24-h incubation in serum-free conditions, fresh DMEM was added (time 0) with 10% charcoal-stripped FBS in the absence or presence of 25-OHD3 (1 µM) and incubations continued for up to 96 h. This assay, which assesses cellular growth on the basis of the intensity of a colorimetric reaction resulting from the reduction of MTT reagent to a soluble formazan salt, was performed as previously described (23). Briefly, 20 µl of the mixture solution were added to each well and incubations continued for 4 h at 37°C. Absorbance at 490 nm was measured using a Bio-Rad microplate reader. Background absorbance was subtracted at each point using a reference absorbance of 700 nm. For cell count, cells were grown in six-well plates in DMEM containing 10% charcoal-stripped FBS until 10% confluency. Following a 24-h incubation in serum-free DMEM, cells were treated as described for the formazan assay, trypsinized at timed intervals, and an aliquot was counted in a Coulter counter (Coulter Electronics, Beds, United Kingdom).

Flow Cytometry
Cells were seeded at a density of 5 x 105 cells into 100-mm plates and grown as described above until 20% confluence. Following a 24-h incubation in serum-free conditions, fresh DMEM was added with 10% charcoal-stripped FBS in the absence or presence of 1 µM 25-OHD3, and incubations were continued. After 24 h, 1 ml of the BrdUrd labeling solution was added to each plate and incubations continued for 1 h at 37°C. Cells were then trypsinized, fixed, denatured, and immunostained with In Situ Cell Proliferation BrdU-Fluos kit according to the manufacturer's protocol (Roche, Mannheim, Germany). Cells were kept on ice for at least 1 h and then analyzed in a FACScan (Becton Dickinson, Mountain View, CA). Calculation of percentage distribution in different phases of the cell cycle was performed with CellQuest software.

Northern Blot Analysis
Polyadenylated mRNA was isolated with the QuickPrep Micro mRNA purification kit according to the manufacturer's's protocol (Amersham Pharmacia Biotech, Baie d'Urfé, QC, Canada) and dissolved in diethylpyrocarbonate-treated water. Twenty µg of mRNA were loaded on a 1.3% agarose formaldehyde gel electrophoresed, transferred to Hybond-N+ nucleic acid transfer membrane (Amersham Pharmacia Biotech), and hybridized with a 340-bp KpnI fragment of the 1{alpha}-hydroxylase cDNA as previously described (39). The same blot was also hybridized with an 86-bp AvaII fragment of human VDR cDNA (40).

Immunocytochemistry
Cells were seeded in six-well cluster plates in DMEM containing 10% charcoal-stripped FBS and grown to ~15% confluency. Following a 24-h incubation in serum-free conditions, fresh medium was added with 10% charcoal-stripped FBS in the absence or presence of 25-OHD3 (1 µM) or 1,25(OH)2D3 (0.1 µM). Incubation was continued for 5 days with a medium change at 2 days. Cells were then fixed in 95% ethanol for 10 min, rinsed with PBS, and kept at 4°C for keratin staining.

Tumors were fixed in 10% neutral formaldehyde buffer and embedded in paraffin. Tumors and cultured cells were then rehydrated and stained by a modification of the three-layer peroxidase/antiperoxidase technique (41), using monoclonal anti-keratin antibody AE3 (DAKO Diagnostics Canada Inc., ON, Canada) (42). As reported earlier, this antibody recognizes keratins with molecular weights of Mr 58,000 and 65,000–67,000. The Mr 65,000–67,000 keratin is regarded as a keratinization marker and easily detectable in the in vivo human epidermis (43).

Generation of Targeting Constructs
Five hundred ng of genomic DNA, prepared from cultured HPK1Aras cells according to a standard protocol, were used to amplify 4 kb of the 1{alpha}-hydroxylase gene by PCR (40 cycles of 92°C, 1 min; 55°C, 2 min; 72°C, 2 min) using HOT TUB DNA polymerase (3.0 units/µl) (Amersham Pharmacia Biotech) in the presence of 1 mM of each dNTP, 2.5 mM MgCl2, 1x reaction buffer, and 500 ng of primers. The primers used were as follows: forward 5'-ATGACCCAGACCCTCAAGTACGCCT-3', and reverse 5'-CTTCCTGAGTCAGGCCAAGTGCATAC-3'. PCR products were analyzed on a 1.5% agarose gel, and compared to molecular weight markers (Life Technologies, Inc.). The 4.0-kb PCR product was then cloned into the pCRII vector using the TA cloning kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Nucleotide sequence of the 1{alpha}-hydroxylase gene was then determined by T7 Sequencing Kit (Amersham Pharmacia Biotech) with [{alpha}-32P]dCTP (Amersham Pharmacia Biotech). DNA and amino acid sequences were analyzed using the DNASTAR (DNASTAR, Inc., Switzerland) and BLAST programs (44). This sequence has been deposited in the GenBank data library under accession no. AF256213. To generate the targeting construct plasmid, the 4-kb PCR 1{alpha}-hydroxylase gene fragment was cloned into the pCRII vector. Subsequently, a 2.2-kb neomycin-resistant cassette flanked by BGH pA and SV40 pA was inserted into the gene between exons 2 and 3 using SmaI and NotI sites causing its disruption (Fig. 1A). Constructs were isolated and purified as described in Maniatis et al. (45).

Generation of KO Cells
Subconfluent (70%) T75 flasks of HPK1Aras were transfected overnight with 2 µg of the targeting plasmid construct or control vector (pcDNA3) using 10 µl of LipofectAMINE (Life Technologies, Inc.) in serum-free DMEM. The medium was then replaced with fresh DMEM containing 10% FBS and 300 µg/ml of G418 (Sigma Aldrich, Oakville, ON, Canada) for 3 weeks. Two hundred eight G418-resistant colonies were obtained and expanded. Thirty-eight individual colonies were then picked and transferred to T75 flasks in DMEM 10% FBS for an additional 3 weeks. These cell lines were then analyzed by Southern blotting to determine the insertion of the targeting construct through this first round of selection. This initial selection permits the selection of heterozygous SKO cells. The heterozygous cell lines generated through this first round of G418 selection were then further selected with higher concentration of G418 (500 µg/ml) for an additional 8 weeks and 20 passages. Individual colonies were then picked and expanded in DMEM 10% FBS as described above and genomic DNA was extracted for Southern blotting to identify DKO homozygous cell lines for the 1{alpha}-hydroxylase gene. WT HPK1Aras cells transfected with the vector alone (pcDNA3) were used as control. Genomic DNA (20 µg) from control, SKO, and DKO HPK1Aras cells was then digested with BamHI and electrophoresed on a 0.8% agarose gel for Southern analysis with a 340-bp NotI/NcoI fragment of the 4.0-kb fragment cloning PCR product. Hybridization was performed with QuikHyb solution (Stratagene, La Jolla, CA), Ready-To-Go kit (Amersham Pharmacia Biotech), and [{alpha}-32P]dCTP (Amersham Pharmacia Biotech) according to the manufacturer's's instructions.

Animal Protocols
Control (WT) or DKO HPK1Aras cells (3 x 106) were suspended in 0.2 ml of the mixture (1:1) of PBS and Matrigel (Becton Dickinson Labware, Franklin Lakes, NJ) and injected subcutaneously in the neck of female SCID mice (Charles River, St. Constant, QC, Canada). Two weeks after tumor implantation, an Alzet osmotic mini-pump (ALZA Corp., Palo Alto, CA) containing 25-OHD3 (2000 pM/24 h) was implanted immediately juxtaposed to the tumor site on the back of the animal. Preliminary experiments determined the dosage of 25-OHD3 concentrations in non-tumor-bearing animals. 3D tumor measurements were done using calipers. Tumor diameter long axis (L) and mean mid-axis width (W) were measured to estimate the tumor volume using the following formula:

Growth curves were generated by plotting the mean tumor volume of mice given injections of control HPK1Aras cells against mice given injections of DKO HPK1Aras cells.

Plasma Calcium, 25-OHD3, and Weight Measurements
Plasma samples were obtained by orbital bleeding at regular intervals and 50–100 µl were used to measure total calcium and albumin by microchemistry (Kodak Ektachrome) and 25-OHD3 by RIA (DiaSorin, Stilwater, MN). Body weight was measured once a week using OHAUS LS200 scale (VWR, Montreal, Canada).

Determination of 1{alpha}-Hydroxylase Activity
WT, SKO, and DKO HPK1Aras cells were seeded at a density of 1.1 x 107 cells into T-160 flasks and grown until 50% confluency in DMEM containing 10% charcoal-stripped FBS. Following a 24-h incubation in serum-free DMEM, the medium was replaced with DMEM containing 1% BSA (Life Technologies, Inc.) and 1 µM 25-OHD3, and incubated for another 6 h. One ml of conditioned medium containing 1000 cpm of [3H]-1,25(OH)2D3 (Amersham, Piscataway, NJ) to monitor recovery through the extraction and chromatographic procedures was extracted utilizing the solid-phase procedure described by Hollis (46) and 1,25(OH)2D3 concentrations determined using a commercially available kit (DiaSorin). In some cases, the semipurified 1,25(OH)2D3 was collected and subjected to HPLC as described by Horst et al. (47). HPLC was performed using a Zorbax Sil column (DuPont Instruments, Wilmington, DE) eluted with hexane:methylene chloride:isopropanol (49:49:2). Aliquots were collected from the HPLC column and 1,25(OH)2D3 concentrations were determined.

Statistical Analysis
All results of in vitro experiments are expressed as the mean ± SE of replicate (at least triplicate) determinations and statistical comparisons based on ANOVA or by the Student t test. A probability value of P < 0.05 was considered to be significant. Statistical significance of the difference between tumor volume between groups was analyzed by Mann-Whitney test for nonparametric samples.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 

This work was presented in part at the 21st Annual Meeting of the American Society for Bone and Mineral Research in St. Louis, MO, in September 1999.

We thank Judy Kremer for critical review of the manuscript.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 Canadian Institutes of Health Research Grant MT10839 (to R.K.). Back

Received March 27, 2002; revised July 15, 2002; accepted August 6, 2002.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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