Relative resistance to 1,25-dihydroxyvitamin D3 in a keratinocyte model of tumor progression.

We have examined the effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on mitogen-stimulated growth and on c-myc proto-oncogene expression in a keratinocyte model of tumor progression. A dose-dependent inhibition of cell growth by 1,25-(OH)2D3 was demonstrated in both established (HPK1A) and malignant (HPK1A-ras) cells. However, this inhibition was observed with the addition of 1,25-(OH)2D3 at a higher concentration in HPK1A-ras cells than in HPK1A cells. Cell cycle analysis revealed a blockage of the normal progression of the cell cycle from G0 to S phase in the presence of 1,25-(OH)2D3. A higher concentration of 1,25-(OH)2D3 was required in HPK1A-ras cells to overcome the mitogen-stimulated progression into S phase, when compared with HPK1A cells. Analysis of c-myc messenger RNA revealed a strong inhibition of its expression at early time points with higher concentrations of 1,25-(OH)2D3 being required to obtain an inhibition in HPK1A-ras cells similar to that obtained in HPK1A cells. 1,25-(OH)2D3 receptor characterization by sucrose gradient analysis and equilibrium binding demonstrated the presence of a single 3.7 S protein with similar receptor numbers and affinity in both cell lines. These observations therefore demonstrate that an alteration of the growth inhibitory response to 1,25-(OH)2D3 occurs when keratinocytes acquire the malignant phenotype and suggest that the alteration lies beyond the interaction of the ligand with its receptor. In addition, relative resistance to 1,25-(OH)2D3 was also observed in the expression of the cell-cycle associated oncogene c-myc. These studies may therefore have important implications in vivo in the development and growth of epithelial cell cancers.

We have examined the effect of 1,225-dihydroxyvitamin D3 (1,2D3) on mitogen-stimulated growth and on c-myc proto-oncogene expression in a keratinocyte model of tumor progression. A dose-dependent inhibition of cell growth by 1,2D3 was demonstrated in both established (HPKlA) and malignant (HPKlA-rw) cells. However, this inhibition was observed with the addition of 1,25-(OH)2D3 at a higher concentration in HPKlA-rus cells than in HPKlA cells. Cell cycle analysis revealed a blockage of the normal progression of the cell cycle from Go to S phase in the presence of 1,25-(OH)2D3. A higher concentration of 1,2D3 was required in HPKlA-ras cells to overcome the mitogen-stimulated progression into S phase, when compared with HPKlA cells. Analysis of c-myc messenger RNA revealed a strong inhibition of its expression at early time points with higher concentrations of 1,25-(OH)2D3 being required to obtain an inhibition in HPKlA-rus cells similar to that obtained in HPKlA cells. 1,25-(OH)2D3 receptor characterization by sucrose gradient analysis and equilibrium binding demonstrated the presence of a single 3.7 S protein with similar receptor numbers and affinity in both cell lines. These observations therefore demonstrate that an alteration of the growth inhibitory response to 1,225-(OH)2D3 occurs when keratinocytes acquire the malignant phenotype and suggest that the alteration lies beyond the interaction of the ligand with its receptor. In addition, relative resistance to 1,25-(OH)2D3 was also observed in the expression of the cell-cycle associated oncogene c-myc. These studies may therefore have important implications in vivo in the development and growth of epithelial cell cancers.
Carcinogenesis is a multistep process (l), and epidemiological studies have suggested that five or six independent steps are required for acquisition of the malignant phenotype (2). One of the mechanisms that has been implicated in neoplastic development is the cooperative action of two or more oncogenes (3) which are thought to act in a positive way to control cell growth in response to known mitogenic factors (4). How-11 To whom correspondence and reprint requests should be addressed: Calcium Research Laboratory, Royal Victoria Hospital, Rm. H4.67, 687 Pine Ave. West, Montreal, Quebec H3A 1A1, Canada. ever, equally important to the tumor development process may be negative regulators of cell growth which oppose the action of known growth factors. 1725-Dihydroxyvitamin D3 (1,2D3)' has been identified recently as an important factor controlling the growth of HL-60 human promyelocytic leukemic cells and their differentiation into the monocytic cell type (5,6). This steroid has also been shown to have antiproliferative capabilities in normal keratinocytes (7,8). Specific receptors for 1,25-(OH)2D3 have been demonstrated in both normal and malignant murine keratinocytes (9) which are thought to mediate the biological effects of the hormone. In addition to its inhibitory action on growth, previous studies have demonstrated an altered expression of specific oncogenes, such as c-myc, in response to 1,25-(OH)2D3 (10,11).' In normal human keratinocytes alterations in growth elicited by 1,25-(OH)2D3 were shown to be accompanied by a rapid inhibition of c-myc (13) expression.' In the present study we have examined the effects of 1,25-(OH)2D3 on growth and cmyc expression in a human keratinocyte model of tumor cell progression (14, 15). In this model primary human foreskin keratinocytes were established as an immortalized cell line after transfection with human papillomavirus type 16 and subsequently transformed by an activated ras oncogene. Using this model system we have studied the effects of factors shown previously to modulate the growth of normal human keratinocytes, such as epidermal growth factor (EGF), fetal bovine serum (FBS), and 1725-(OH)2D3.

MATERIALS AND METHODS
Culture of Human Keratinocyte Cell Lines-The HPKlA cell line was established from normal human keratinocytes by stable transfection with human papillomavirus type 16 (14). Despite acquiring an indefinite life span in culture these cells retain differentiation properties characteristic of normal keratinocytes (16) and are nontumorigenic when injected into nude mice (Table I). These immortalized cells were subsequently transformed into the malignant HPKlA-ras cell line after transfection with a plasmid carrying an activated Hras oncogene (15). In addition to forming colonies in soft agar the malignant HPKlA-ras cells produce invasive squamous cell carcinoma when transplanted into nude mice (Table I). Both immortalized (HPKlA) and malignant (HPKlA-ras) cell lines were seeded and grown in Dulbecco's modified Eagle's medium (DMEM) (GIBCO) supplemented with 10% FBS (GIBCO) and passaged once or twice weekly.
XTT-Microculture Tetrazolium Assay for Cell Growth-This assay, performed as described previously (17)  Thymidine (1 pCi/ml; Du Pont-New England Nuclear) was added to cultured cells during the last 2 h of incubation. After aspiration of the medium cells were washed twice with cold Hanks' balanced salt solution and then incubated in 5% cold trichloroacetic acid for 15 min. After aspiration of the trichloroacetic acid, the cell layers were dissolved in 1 ml of 0.6 N NaOH and an aliquot removed for liquid scintillation counting. Triplicate wells/plate were trypsinized and aliquots counted to correct the [3H]thymidine cpm for cell number.
Results were then expressed as percent of EGF-stimulated activity.
Flow Cytornetry-Cells were seeded at a density of lo5 and 5 X lo' cells/well for HPKlA and HPKlA-ras, respectively, in six-well cluster plates and grown to 20% confluence. After a 24-h period in basal conditions fresh DMEM supplemented with 10 ng/ml EGF and varying concentrations of 1,25-(OH)~& was added to the cultures for 24 h. After trypsinization, cells were centrifuged at low speed (600 X g), rinsed once with phosphate-buffered saline, and stained according to the technique described by Vindeldv (18). The pellet was resuspended in 1 ml of the following solution added dropwise while vortexing: 3.5 mM Tris, 7.5 p~ propidium iodide (Calbiochem), 0.1% Nonidet P-40 (Sigma), 700 units/liter RNase (Boehringer Mannheim), and 10 mM NaCl. After standing at least 10 min on ice, the nuclei were analyzed in a FACScan (Becton Dickinson Inc., Oxnard CA). Calculation of percentage distribution in different phases of the cell cycle was performed with CellFit software (Becton Dickinson Inc.) using a sum of broadened rectangles fit. RNA Analysis-Cells were plated in the same manner as for flow cytometry studies, in DMEM supplemented with 10% FBS, trypsinized at timed intervals, centrifuged at low speed (600 X g), rinsed with phosphate-buffered saline, and lysed with a mixture of 4 M guanidine thiocyanate, 25 mM trisodium citrate, 1 mM EDTA, and 0.1 M 0-mercaptoethanol (GTC mixture). GTC extracts were stored a t -70 "C for subsequent RNA analysis by dot blot hybridization or Northern blot hybridization.
For Northern Blot analysis, GTC extracts were purified by cesium chloride gradient ultracentrifugation (19) and 10 pg of total RNA electrophoresed on a 1.1% agarose-formaldehyde gel. Ethidium bromide was added to each sample before electrophoresis to permit detection by UV transillumination and to ensure that equivalent quantities of RNA were loaded into all lanes. RNA was transferred by blotting to a nylon membrane (Nytran). For dot blot hybridization, samples were processed as described earlier (10). The filters were airdried, baked at 80 'C for 2 h, and then hybridized with a ClaI-EcoRI restriction fragment encoding exon 111 of the human c-myc gene (20). This probe was labeled with [32P]dCTP (ICN Biomedicals of Canada Ltd., Mississauga, ON) using a random primer kit (Amersham Corp.).
After incubation at 42 "C for 24 h, filters were washed twice at room temperature for 30 min each in 2 X standard saline citrate (SSC), 0.1% sodium dodecyl sulfate (1 X SSC is 0.15 M sodium chloride, 0.015 M trisodium citrate), and then twice for 30 min each in 0.5 X SSC, 0.1% sodium dodecyl sulfate at 55 "C. Autoradiography of filters was carried out at -70 "C using Kodak XAR films and two intensifying screens. The intensity of the dot blots was analyzed by laser densitometry (Ultroscan XL, LKB instruments Inc.). Filters were also probed with an 800-base pair BamHI restriction fragment of rat cyclophilin as a control for c-myc mRNA changes.
1,25-Dihydroxyuitamin D3 Receptor Characterization-Binding studies were performed according to a previously published method (21). Confluent cells were scraped and suspended in 1 ml of a buffer containing 300 mM/liter KCl, 10 mM/liter Tris, 1 mM/liter EDTA, 5 mM/liter dithiothreitol, and 10 mM/liter sodium molybdate, pH 7.4 (KTEDM). Cells were sonicated in the buffer for 5 X 30-s intervals on ice at maximum setting using a Brinkmann sonicator and centrifuged at 80,000 X g for 60 min at 4 "C. 180 pl of cytosol adjusted to contain 500 pg of protein containing vitamin D receptors (VDR) was incubated with 10,000 cpm of 3H-labeled 1,25-(OH)2D3 (Du Pont-New England Nuclear) in 20 pl of ethanol and increasing concentrations of unlabeled 1,25-(OH)2D3 for 16 h at 4 "C. Nonspecific binding was assessed in the presence of 200-fold excess nonradioactive 1,25-(OH)2D3. Bound and unbound hormone were separated using 100 p1 of dextran-coated charcoal (0.15% dextran and 1.5% charcoal in KTEDM). After 15 min of incubation followed by centrifugation, 100 p1 of the supernatant was counted by liquid scintillation spectroscopy.
Sucrose density gradient analysis was performed using a linear 4-20% gradient of sucrose solutions in KTEDM buffer. 180 p1 of cytosol was incubated for 16 h at 4 "C with 20 pl of ethanol containing 10,000 cpm of 3H-labeled 1,25-(OH)2D3 with or without excess ~,~E J -( O H )~D~ (60 nM). After removal of unbound hormone, with dextran-coated charcoal, supernatants were layered on top of gradients and centrifuged at 257,000 X g for 18 h at 4 "C in a Beckman ultracentrifuge. The radioactivity in 0.1-ml fractions was determined by liquid scintillation spectroscopy.
Statistical Analysis-Statistical significance was determined by one-way analysis of variance or by Student's t test, and results were expressed as mean f S.E. of replicate determinations. A probability value of < 0.01 was considered to be significant.    Dot Blot Analysis-Total cellular RNA extracted from equal numbers of HPKlA and HPKlA-rm cells at timed intervals, after stimulation with 10% FBS, revealed a rapid and transient induction of c-myc mRNA in both cell lines. When expressed as fold stimulation above basal, the induction was greater in HPKlA than in HPKlA-rm cultures (Fig. 3). Dose-dependent decreases in this FBS-stimulated activity in response to 1,25-(OH)2D3 were noted in both cell lines, although the inhibitory response was greater in the HPKlA than in HPKlA-rm cells (Fig. 4).

Effects of 1,25-(oH)2D3
Northern Analysis-Northern analysis of total RNA removed at timed intervals from HPKlA and HPKlA-rm cells cultured in the absence or presence of M 1,25-(OH)& revealed one major transcript of 2.4 kilobases in both cell lines (Fig. 5). The addition of 10% FBS resulted in a stimulation of c-myc mRNA expression which was greater in HPKlA (panel A ) than in HPKlA-rm (panel B). Although  (27). The myc gene product is thought to mediate a signal associated with cell division and appears to be required for normal cell growth (28). The rapid induction of c-myc after stimulation by agents such as growth factors and serum (4,29) suggests a potential role for the protein in the passage of cells from the resting phase into the actively dividing phase of the cell cycle.
In the present study, a characteristic time course of c-myc mRNA expression in response to stimulation by mitogens was observed in both cell lines with an early peak and a slow decline over 24-48 h. However, maximum levels of induction were greater in HPKlA than in HPKlA-ras cells. The apparent independence, exhibited by the malignant cells, from exogenous growth promoters could be a function of overproduction of endogenous transforming growth factor-m (30), constitutively active EGF receptors (31), or altered positive responsive elements located in the promoter region of the cmyc gene (32).
In previous studies, we and others have shown that expression of c-myc mRNA was negatively regulated by 1,25-(OH)& in several cell types (10)(11)(12)(13)33). In addition, 1,25-(OH)zD3 has been shown to regulate c-myc gene expression at the transcriptional level (33). In the present study, the immortalized HPKlA cells showed a strong inhibition of cmyc mRNA expression similar to that described previously in normal keratinocytes in response to lo-' M 1,25-(OH)2D3.2 The neoplastic HPKlA-ras cells, on the other hand, responded to a 10-fold greater dose with minimal inhibition of serum-stimulated c-myc mRNA levels at early time points and with an apparent rebound at 24 h.
We have noted previously the same type of altered response at the mRNA level of another 1,25-(OH)2D3-responsive gene, the parathyroid hormone-related peptide (PTHrP) gene, in the HPKlA-ras keratinocytes (34), as compared with normal human keratinocytes (35). This relative resistance could occur at the level of receptor-ligand interaction as a function of altered intracellular metabolism of the steroid, altered receptor function or a constitutional absence, or decreased expression of the 1,25-(OH)zD3 receptor in the malignant cells. Soluble extracts from HPKlA and HPKlA-ras cells demonstrated similar 1,25-(OH)2D3 binding characteristics including receptor number, receptor affinity, and migration pattern of the receptor-ligand complex on a sucrose density gradient which were similar to those found in normal human keratinocytes (36). Analysis of medium conditioned by HPKlA and HPKlA-ras cells for 48 h in the presence of 1,25-(OH)2D3 showed no significant difference in the levels of the steroid (data not shown). Taken together, these observations suggest that the relative resistance to 1,25-(OH)zD3 demonstrated in the malignant keratinocytes in these studies lies somewhere beyond the interaction of the ligand with its cytosolic receptor.
The mechanism by which 1,25-(OH)2D3 suppresses gene activity remains elusive. One potential mechanism could involve direct suppression via binding of the 1,25-(OH)zD3receptor complex to as yet unidentified responsive elements in target genes such as c-myc. An alternative mechanism could involve competition between the VDR and other transactivating factors which normally regulate transcriptional activity of that gene (37). Both positive and negative regulation of transcription by thyroid hormone receptor and retinoic acid receptor heterodimers has been demonstrated (38). In this context competition between positive regulators of c-myc expression, such as growth factors or serum, and the 1,25-(OH)zD3-receptor complex could be altered in the ras-transformed keratinocytes. Perhaps an excess of unidentified positive regulatory factors present in the HPKlA-ras cells confers a requirement for an increased concentration of 1,25-(OH)zD3 to form sufficient 1,25-(OH)zD3-receptor complexes to compete effectively for binding to the genetic regulatory element. Inasmuch as repression of c-myc transcriptional activity was measured in the present studies in the presence of serum this could prove to be a plausible hypothesis. Identification of a serum-responsive element, a vitamin D-responsive element (VDRE), and other regulatory factors as well as definition of their interactions with one another will be required to clarify this issue.
It has been suggested that binding of 1,25-(OH)~D3 to its receptor induces a conformational change allowing for receptor phosphorylation (39). In addition, serine 51 of the VDR has been identified as an effective substrate for protein kinase C-p and the resulting phosphoprotein shown to be important to VDR-mediated transcription (40). An anomalous phosphorylation process in the ras-transformed cells could perhaps contribute to the 1,25-(OH)zD3 relative resistance demonstrated by these cells. Additionally a sequence between amino acids 102 and 112 outside of the DNA binding domain, in the VDR, has been postulated by Haussler et al. (37) to be necessary in the nuclear transfer of the receptor and could therefore constitute another potential site of inactivation in the rus-transformed cells. Alternative possibilities could encompass altered interactions between VDR and cis-acting VDREs and/or between VDR and other trans-activating factors necessary for transcriptional regulation (41) and/or for stabilization of the VDR/VDRE (12). Thus, the apparent transience of the inhibitory action of 1,25-(OH)zD3 in the malignant HPKlA-ras cells could be a function of an unstable VDR/VDRE association. Identification of both a consensus VDRE as well as accessory proteins involved in the interaction between the VDR and its target genes in these cells will be required to clarify these issues.
The preceding studies therefore demonstrate that resistance to an important negative regulator of keratinocyte cell growth is acquired in the passage from established to malignant phenotype. These findings may have important implications in vivo in the development and unrestrained growth of squamous carcinomas.