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Carcinogenesis, Vol. 22, No. 12, 1955-1963, December 2001
© 2001 Oxford University Press

CANCER BIOLOGY

Overexpression of retinoic acid receptors alpha and gamma into neoplastic epidermal cells causes retinoic acid-induced growth arrest and apoptosis

Asma Hatoum1, Marwan Eid El-Sabban2, Joe Khoury1, Stuart H. Yuspa3 and Nadine Darwiche1,4,5

1 Departments of Biochemistry,
2 Human Morphology,
4 Biology, American University of Beirut, Beirut, Lebanon; and
3 Laboratory of Cellular Carcinogenesis and Tumor Promotion, Division of Basic Science, National Cancer Institute, NIH, Bethesda, MD 20892, USA


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Retinoids are essential for normal epidermal differentiation and are used for the prevention and treatment of numerous skin disorders and cancers in humans. In previous studies, we have shown that retinoic acid receptors (RARs) -{alpha} and -{gamma} are down-regulated during skin tumor progression. The transduction of v-rasHa into primary mouse keratinocytes is sufficient to reduce both RAR{alpha} and RAR{gamma} protein levels as well as inhibit their transactivation functions. Our primary objective is to investigate the roles that RAR{alpha} and RAR{gamma} play in keratinocyte tumor cell proliferation. Through retroviral gene transduction, we overexpressed RAR{alpha} or RAR{gamma} into neoplastic mouse epidermal cells with down-regulated endogenous RAR proteins. Following all-trans retinoic acid (RA) treatment, RAR{alpha}- and RAR{gamma}-transduced cell lines exhibit a progressive, dose-dependent growth inhibition relative to the control LXSN cell lines. Further characterization of RAR-transduced cells following RA treatment reveals that both RAR{alpha} and RAR{gamma} cause a decrease in S-phase population, while only RAR{alpha} causes a simultaneous G0/G1 block as evidenced by reduced [3H]-thymidine incorporation and flow cytometric analysis of DNA content. Following RA treatment, both receptors cause an early, transient increase in the cyclin-dependent kinase inhibitor (CDKI) p21 proteins, while only RAR{alpha} causes a simultaneous sharp, brief increase in the CDKI p16 protein. A later decrease in cyclin D1 protein is also evident in RAR{alpha}- and RAR{gamma}-transduced cells. Chromatin condensation and PARP cleavage are observed in both RAR{alpha}- and RAR{gamma}-transduced cells indicating an RA-induced apoptosis that may be caspase dependent. Furthermore, both receptors cause a late upregulation and apparent cleavage of the squamous differentiation marker protein kinase C (PKC)-{eta}. These results suggest that RAR{alpha} and RAR{gamma} enhance growth suppression and apoptosis of neoplastic epidermal keratinocytes. This growth inhibitory effect of both retinoid receptors in neoplastic keratinocytes may be achieved through distinct as well as overlapping mechanisms of cell cycle control.

Abbreviations: CDKI, cyclin-dependent kinase inhibitor; DMSO, dimethylsulfoxide; K, keratin; PKC, protein kinase C; RA, all-trans-retinoic acid; 9c-RA, 9-cis-retinoic acid; RAR, retinoic acid receptor; RARE, retinoic acid response element; RXR, retinoid X receptor


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Retinoids are essential for multiple key physiological processes including reproduction, embryonic development, epithelial differentiation, immune function, and vision (1,2). Ever since the discovery of the retinoid receptors, a growing interest in the molecular mechanisms generating these retinoid-induced pleiotropic effects has marked the past decade of retinoid research (3). Retinoids regulate epidermal cell growth and differentiation (4–7), and are therefore commonly used for the treatment of various skin disorders including psoriasis (8), acne (4), and cancer (9–11). Although the use of retinoids as differentiating and chemopreventive agents has been well established in the clinical setting (4,8,11), much remains to be discovered concerning the diverse retinoid signaling pathways in growth and differentiation.

Retinoid receptors are members of the steroid/thyroid hormone nuclear receptor superfamily and fall into two major categories—the retinoic acid receptors (RARs), and the retinoid X receptors (RXRs) (2,3). Three genes, designated as {alpha}, ß, and {gamma}, code for RARs and RXRs and produce numerous isoforms of each through differential splicing and the use of alternative promoters. After binding all-trans retinoic acid (RA) or 9-cis retinoic acid (9c-RA), RARs heterodimerize with RXRs and exert transcriptional regulation of target genes (12). The RXR physiological ligand is 9c-RA, and RXRs act as transcriptional factors through homodimerization as well as heterodimerization with other members of the same superfamily including thyroid hormone and vitamin D3 receptors (13).

Retinoid receptors are differentially expressed in embryonic and adult tissues. In the epidermis, the whole of retinoid signaling is carried out by RARs -{alpha} and -{gamma} and RXRs -{alpha} and -ß (14,15). Epidermal RXR protein levels far exceed RAR levels, and of the two families, RXR{alpha} and RAR{gamma} are the most abundantly expressed in both human (14) and mouse (15) epidermis.

Retinoid receptor signaling pathways are aberrant or disrupted during the neoplastic process and vary according to the different cancers (16). Exogenous expression of different retinoid receptors, mostly RARs, increased retinoid responsiveness in leukemic (17) and teratocarcinoma cells (18), and lung (19,20), head and neck (21,22), breast (23) and ovarian cancerous cells (24). Our previous studies using the two-stage model of mouse epidermal carcinogenesis (25) reveal that RAR{alpha} and RAR{gamma} are down-regulated during skin tumor progression, while RXR levels remain relatively unchanged (15). In addition, initiation of primary keratinocytes through v-rasHa oncogene transduction is sufficient to reduce epidermal RAR proteins (26). Interestingly, chemically-induced papillomas with a high risk for premalignant progression express much lower levels of RAR proteins and are therefore less responsive to RA treatment than low-risk papillomas (27). Progressive decreases in nuclear retinoid receptors were also recently observed during squamous carcinogenesis in human (28).

When applied to normal skin, RA causes hyperplasia and suppression of numerous terminal differentiation markers including the keratin pair K1/K10 expression and cornified envelope formation (4,6). In contrast, RA applied to skin tumors in the early stages of development causes tumor growth inhibition and reduced incidence of malignant transformation (5,9,27,29,30). This modulatory effect of retinoids on slow and rapidly-growing keratinocytes implicates retinoid receptors as substantial differential regulators of normal as well as neoplastic epidermal cell homeostasis. Although some cell cycle regulators in various malignancies have been identified in retinoid-induced growth inhibition such as p21 (31), p27 (32), and cyclin D1 (33), the molecular mechanisms through which retinoids exert their cell cycle control in neoplastic epidermis remain largely uncharacterized.

In the present study, we generated mouse epidermal cell lines that overexpress either RAR{alpha} or RAR{gamma} in order to determine their role in neoplastic epidermal cell growth and untangle their differential signaling pathways. Even though RARß has been implicated in growth inhibition of lung cancer cells (19) and in head and neck cancers (21) and its decreased expression has been linked to retinoid resistance, we did not exogenously introduce this receptor in epidermal cell lines because RARß is not endogenously expressed in primary epidermal keratinocytes or epidermis (14,28,34). Although both RAR{alpha} and RAR{gamma} seem to elicit a similar retinoid-induced growth inhibition and apoptosis in transduced neoplastic epidermal cells, each receptor appears to mediate these effects through distinct as well as overlapping pathways.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell culture
The neoplastic SP-1 and 308 papilloma cell lines were produced in SENCAR and BALB/c mice, respectively, as described previously (35,36). Both cell lines carry an activating mutation in the c-rasHa oncogene and produce benign dysplastic papillomas when grafted onto the dorsum of immune-deficient mice (35). All keratinocytes were cultured in Spinners minimal essential medium (Gibco, Bethesda, MD) containing 9% dialysed fetal bovine serum (Gibco) and 0.05 mM Ca2+ to maintain a basal cell phenotype (37), and cells were used at 50–60% confluency. All-trans retinoic acid was obtained from Sigma Chemical Co. (St Louis, MO), diluted in dimethylsulfoxide (DMSO) under amber light, and stored at –80°C.

Retroviral infection of cells
The retroviral vectors LRARSN, in which the full length RAR{alpha} or RAR{gamma} cDNA is introduced into the retroviral vector LXSN (38), were generously provided by Steven J.Collins (Fred Hutchinson Cancer Research Center, Seattle, Washington). SP-1 and 308 cells were infected with LXSN, LRAR{alpha}SN, or LRAR{gamma}SN retroviral vectors in the presence of 4 µg/ml polybrene (Sigma). Infectants were selected in growth medium supplemented with G418 as described (38), except that 25 µg/ml were used.

Immunoblot assays
Cellular protein extracts were prepared from cultured attached or detached keratinocytes, washed twice with ice-cold PBS, and scraped either into SDS-lysis buffer (0.25 M Tris–HCl pH 6.8, 20% glycerol, 4% SDS, 0.002% bromophenol blue, 10% ß-mercaptoethanol) or PARP extraction buffer (62.5 mM Tris, pH 6.8, 6 M urea, 10% glycerol, 2% SDS, 0.003% bromophenol blue, 5% ß-mercaptoethanol) as indicated in the figure legends. Protein concentration was determined using the DC Protein Assay from Bio-Rad (Hercules, CA). Cellular protein extracts (25–50 µg) were run on denaturing 10–12% polyacrylamide gels and transferred onto nitrocellulose membranes (Bio-Rad) by standard blotting procedures. RAR{alpha}, RAR{gamma}, RXR{alpha}, p16, cyclin D1 and PKC{eta} polyclonal antibodies were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and were used at a dilution of 1:1000. Polyclonal antibodies against p21 (Santa Cruz and Oncogene Research Products, Cambridge, MA), and PARP (Enzymes Systems Products, Livermore, CA) were used at dilutions of 1:500 and 1:5000, respectively. Equal protein loading and quality was verified through keratin 14 (39) reprobing of membranes and parallel protein gel Coomassie staining. Proteins were detected by enhanced chemiluminescence using the ECL system (Santa Cruz) with horseradish peroxidase-conjugated secondary antibody (Santa Cruz) at a 1:5000 dilution.

Electrophoretic mobility shift assay
For nuclear extract preparation, cells were washed twice in ice-cold PBS and frozen at –80°C. Cells were then scraped off the plates in ice-cold buffer A (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, and 1 mM dithiothreitol (DTT)). The nuclei were then pelleted and lysed in buffer C (20 mM HEPES, 400 mM NaCl, 1.5 mM MgCl2, 0.2 mM ethylenediaminetetraacetic acid (EDTA), 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 25% glycerol). Finally, the nuclei were extracted by gentle mixing for 30 min and pelleted. The supernatant was diluted with buffer D (20 mM HEPES, 50 mM KCl, 20% glycerol, 0.2 mM EDTA, 1 mM DTT, and 0.5 mM PMSF) and stored at –80°C. Protein concentration was determined using the DC Protein Assay (Bio-Rad). ß-retinoic acid responsive element (ß-RARE) consensus oligonucleotide (Santa Cruz) was end-labeled with {gamma}-32P adenosine triphosphate using T4 polynucleotide kinase. Nuclear proteins (10 µg) and labeled probe (0.4 ng, >30 000 c.p.m.) were run on 5% non-denaturing polyacrylamide gels. The gels were dried on Whatman filter paper and processed for autoradiography at –80°C. Specificity of RAR binding was assessed by competition experiments using 10-fold excess unlabeled or mutant ß-RARE.

Growth assay
SP-1 and 308 infected cells were seeded into 96-well plates at a density of 2500 cells per well. At 50–60% confluency, the cells were treated with 0.1% DMSO or varying concentrations of RA (3 x 10–10-3 x 10-6M). Medium was replenished every second day, and viability after 2, 4, or 5 days post-RA treatment was assayed using the CellTiter 96® non-radioactive cell proliferation assay kit as suggested by the manufacturer (Promega Corp., Madison, WI). Results are expressed as growth relative to DMSO-treated controls and are derived from the mean of quadruplicate wells.

[3H]-Thymidine incorporation
To assay the effect of expression of exogenous RAR{alpha} or RAR{gamma} on cell proliferation, RAR{alpha}, RAR{gamma}, or LXSN-cells were plated in 12-well Falcon tissue culture trays at 5 x 104 cells per well. Cells were treated with varying concentrations of RA (3 x 10-9 to 3 x 10-6 M) or 0.1% DMSO for 48 h and pulsed for 4 h with 0.5 µCi of [3H]-thymidine (Amersham Pharmacia Biotech, Cleveland, OH). The amount of label incorporated into DNA was determined as previously described (40). Triplicate wells seeded in parallel were used to determine total cell number, and the radioactivity associated with individual samples was reported as c.p.m./cell number.

Cell cycle analysis
Asynchronously growing RAR{alpha}, RAR{gamma}, and LXSN SP-1 cells plated in 100 mm Falcon tissue culture dishes were treated for 48 h with 3 x 10-6 M RA. Attached cells were trypsinized, pooled with the detached population, fixed in 80% ethanol, and stored at –20°C for further analysis. Subsequently, cells were incubated for 1 h in 200 µg/ml RNase A and then stained with 50 µg/ml propidium iodide (Sigma). Cell cycle analysis was performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA), and the percentage of cells in pre-G0, G0/G1, S, and G2/M phases was determined using CellQuest analysis program (Becton-Dickinson).

Hoechst nuclear staining
RAR{alpha}, RAR{gamma}, and LXSN SP-1 cells were grown on uncoated glass coverslips in 6-well Falcon tissue culture trays. After 48 h and 72 h of RA treatment (3 x 10-6 M), attached and detached cells were stained with 2 mg/ml Hoechst 33342 nuclear stain (Molecular Probes Inc., Eugene, OR) and fixed in 2% formaldehyde/5% glycerol solution. Detached cells were resuspended in antifade (Molecular Probes Inc.) and mounted on their respective detached population. In addition, slides containing either attached or detached cells were prepared and analyzed separately.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Generation of RAR{alpha}- and RAR-transduced epidermal cell lines
Retroviral infection of either RAR{alpha} or RAR{gamma} genes clearly results in overexpression of respective RAR proteins in SP-1 cell lines as compared with LXSN control cells and primary epidermal keratinocytes (Figure 1AGo). Note that the corresponding SP-1 uninfected and LXSN retroviral control cells barely show any detectable levels of RAR{alpha} and RAR{gamma} proteins, and that all cells retain endogenous levels of RXR{alpha} proteins. These transduced receptors in SP-1 cells are capable of binding to a ß-RARE as evidenced by the gel shift mobility assay (Figure 1BGo). The same results are observed with the 308 cells (data not shown).



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  		Fig. 1. (A) RAR{alpha}- and RAR{gamma}-transduced SP-1 cell lines overexpress the respective receptor proteins. Total SDS cell lysates (30 mg) were examined by immunoblot analysis using antibodies specific for RAR{alpha}, RAR{gamma}, and RXR{alpha}. Blots were stripped and re-probed with K14 to ensure equal loading. (B) RAR binding activity is observed in RAR-infected keratinocytes. Electrophoretic mobility shift assay reveals DNA binding of RAR{alpha} and RAR{gamma} onto a ß-RARE oligonucleotide. Nuclear extracts (10 mg) were run in the presence of labeled probe on a 5% polyacrylamide gel as described. Competition of binding was observed with the addition of 10-fold excess cold probe (C), but not with the addition of 10-fold excess mutant probe (M).

 
Decreased cell proliferation and altered cell cycle distribution of epidermal cells transduced with RAR{alpha} and RAR{gamma}
Before RA treatment, neither RAR{alpha} nor RAR{gamma} cause visible morphological changes in the benign neoplastic SP-1 and 308 cells when compared with LXSN cells (Figure 2Go). However, upon RA treatment, RAR{alpha}- and RAR{gamma}-transduced SP-1 cells exhibit a dendritic morphology and increased internal granularity. The effects of RA treatment on 308 cell morphology is less evident, as RAR{alpha}- and RAR{gamma}-transduced 308 cells appear more circular in shape with few dendritic projections and demonstrate an impaired ability to become confluent post-RA treatment when compared with treated 308 LXSN cells (data not shown).



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  		Fig. 2. RA treatment of RAR{alpha} and RAR{gamma}-transduced SP-1 cells alters cell morphology. Cells were plated on 60 mm Falcon tissue culture dishes at a density of 5 x 104 cells/ml. At 50–60% confluency, cells were treated with DMSO or 3 x 10-6 M RA for 48 h. Cells were then washed in PBS and fixed in a 4% formaldehyde solution. Following RA treatment, both RAR{alpha}- and RAR{gamma}-transduced SP-1 cells are sparse and exhibit a dendritic morphology and internal granularity that is not observed in control LXSN-SP-1 cells.

 
Long-term RA treatment of RAR{alpha} and RAR{gamma} SP-1 keratinocytes reveals a dose-dependent, progressive decrease in viability as compared with LXSN control cells (Figure 3Go). In contrast to normal primary keratinocytes which respond to RA by 2.5-fold stimulation of thymidine incorporation by 48 h (data not shown), RAR{alpha}- and RAR{gamma}-transduced cells begin to show sensitivity to even subpharmacological levels (3 x 10-8 M) of RA as early as 2 days of treatment. However, control SP-1 LXSN cells first begin to show a similar sensitivity by day 5 at ten-fold higher RA concentrations. RAR{alpha}-transduced SP-1 cell lines appear more sensitive to RA than their RAR{gamma}-transduced counterparts. Both RAR{alpha}- and RAR{gamma}-transduced 308 cells do exhibit an increased sensitivity to RA when compared with 308-LXSN cells (data not shown). However, we continued the remainder of our studies on the SP-1 cells because they are better characterized. Next we investigated whether RAR{alpha} or RAR{gamma} cause a redistribution of cells within the cell cycle upon RA treatment.



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  		Fig. 3. Retinoic acid treatment of RAR-transduced neoplastic keratinocytes causes a concentration and time-dependent growth suppression. Note that the control LXSN-SP-1 cells only respond to the highest doses of RA treatment by day 5. Cells were plated in 96-well plates (2500 cells/well). At 50–60% confluency, keratinocytes were treated with DMSO or RA (from 3 x 10-10 to 3 x 10-6 M) for up to 5 days. The plots indicate growth of control LXSN- and RAR{alpha}- and RAR{gamma}-transduced SP-1 cells over a period of 5 days. Viability was assayed as described in Materials and methods. Results are expressed as a percentage of DMSO-treated cells for each group. The values depict averages (± SD) of quadruplicate measurements. Results are representative of at least two independent experiments.

 
Both RAR{alpha} and RAR{gamma} cause a dose-dependent decrease in [3H]-thymidine incorporation 48 h post-RA treatment (from 3 x 10-9 to 3 x 10-6 M) with a 60–70% decrease in proliferation observed at 3 x 10-6 M (Figure 4AGo). At this highest RA dose only a 20% inhibition of 3H-thymidine incorporation was observed in LXSN-SP1 cells. DNA content analysis by flow cytometry reveals an expected decrease in S-phase population up to 50% following 48 h of RA treatment in both RAR{alpha}- and RAR{gamma}- transduced SP-1 cells (Figure 4BGo). While RAR{alpha} causes an accumulation of cells at G0/G1 simultaneous to the decrease in S-phase population, RAR{gamma} does not seem to elicit a G0/G1 cell cycle arrest.




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  		Fig. 4. Retinoic acid treatment of RAR-transduced SP-1 neoplastic keratinocytes causes a decrease in cells in the S-phase of the cell cycle. (A) [3H]-Thymidine incorporation of RAR-transduced SP-1 cells reveals a dose-dependent decrease in S-phase population following RA treatment. Cells were plated in 12-well plates at a density of 5 x 104 cells/ml. At 50–60% confluency, keratinocytes were treated with DMSO or RA (from 3 x 10-9 to 3 x 10-6) for 48 h. Then cells were pulsed for 4 h with 0.5 mCi [3H]-thymidine as described in Materials and methods. The radioactivity associated with individual samples is reported as c.p.m./cell number. [3H]-thymidine incorporation is shown as a percentage of DMSO-treated cells for each group. The values represent an average of two independent experiments ± SEM. (B) The different keratinocytes were treated with 3 x 10-6 M RA for 48 h, stained with PI, and DNA content quantified by flow cytometry. The distribution of cells in the pre-G0, G0/G1, S, and G2/M of the cell cycle was determined using the CellQuest analysis program (Becton-Dickinson). Results are representative of three independent experiments.

 
RAR modulation of cell cycle regulators
In order to understand RAR{alpha}- and RAR{gamma}-mediated signaling pathways in RA induced neoplastic epidermal cell growth inhibition, specific cell cycle protein mediators were monitored at several time points following RA treatment. Our observed G0/G1 cell cycle arrest in RAR{alpha}-transduced cells prompted an investigation of cell cycle mediators specifically involved in the G0/G1 phase of the cell cycle.

The cyclin dependent kinase inhibitors (CDKIs) p21 and p16 proteins bind and antagonize cyclin/cdk complexes in order to halt cell cycle progression. In both RAR{alpha}- and RAR{gamma}-transduced keratinocytes, p21 protein levels sharply increase 2–8 h following RA treatment, and then decrease to basal levels by 12 h (Figure 5B and CGo). The increase in p21 protein was more dramatic in RAR{alpha}-SP-1 cells. In addition to p21 induction, only RAR{alpha} cells display a similar sharp, transient increase in CDKI p16 protein following 2–4 h post-RA treatment (Figure 5BGo).



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  		Fig. 5. Retinoic acid treatment of RAR-transduced and control neoplastic keratinocytes results in differential expression of cell cycle regulators. Cells were plated in 100 mm tissue culture dishes at a density of 5 x 104 cells/ml. At 50–60% confluency, the cells were treated with 3 x 10-6 M RA for 2, 4, 8, 12, 24, and 48 h. Whole cell protein lysates from LXSN-SP-1 (A), RAR{alpha}-SP-1 (B), and RAR{gamma}-SP-1 (C) cells were then immunoblotted with either p21, p16, or cyclin D1 antibodies. All blots were stripped and re-probed with K14 to ensure equal loading. Similar trends in protein levels were observed in two independent experiments.

 
No changes in p21 and p16 proteins were observed within the RA-treated control cells. Furthermore, neither p21 nor p16 proteins were detected in the detached population of all tested cell lines (data not shown).

Cyclin D proteins are expressed during the G1 phase of the cell cycle and cyclin D/cdk4,6 complexes are required for G1 progression. All tested cells express high levels of endogenous cyclin D1 proteins. In both RAR{alpha} and RAR{gamma} cells, cyclin D1 protein levels are significantly down-regulated by 48 h following RA treatment as compared with the LXSN control cells (Figure 5Go). Furthermore, RAR{alpha}-induced cyclin D1 reduction upon RA treatment seems to be more pronounced than that observed in RAR{gamma}-keratinocytes. Again, neither SP-1 cells harbor significant amounts of cyclin D1 proteins in the detached population.

Retinoic acid mediated apoptosis and differentiation in RAR{alpha}- and RAR{gamma}-transduced cells
Next we wanted to determine whether RA-mediated growth inhibition of RAR-transduced cells also results in apoptosis. Through Hoechst staining, we have qualitatively assessed that 48 h RA treatment is sufficient to induce chromatin condensation of RAR-transduced cells (Figure 6AGo). Interestingly, the attached population exhibits partial chromatin condensation, and complete condensation is seen only in the detached population of keratinocytes. In general, at least ten-fold less cells detached in the RA-treated LXSN cells compared with the RAR-infected keratinocytes (results not shown). In addition, PARP cleavage is observed by 72 h following RA treatment in both RAR{alpha}- and RAR{gamma}-transduced cells (Figure 6BGo). Again, this cleavage occurs only in the detached population of keratinocytes. In general, there was not enough proteins collected from RA-treated LXSN cells at 48 h and 72 h. Occasionally when sufficient amounts of proteins were extracted 72 h post-RA-treatment of LXSN-cells, PARP cleavage was also observed in the detached population of keratinocytes (data not shown). These data suggest that RA induces apoptosis mostly in RAR{alpha} and RAR{gamma} transduced neoplastic keratinocytes that may be caspase-dependent.




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  		Fig. 6. Retinoic acid treatment induces apoptosis in RAR-transduced neoplastic keratinocytes. (A) Cells were grown on uncoated glass coverslips and treated with 3 x 10-6 M RA or vehicle DMSO when 50–60% confluent. After 48 h treatment, attached and detached cells were stained with Hoechst, pooled, and mounted in antifade as described in Materials and methods. Hoechst staining reveals chromatin condensation in RAR-transduced SP-1 cells following RA treatment mostly in detached keratinocytes. These cells display a pattern of condensed and fragmented nuclei characteristic of apoptosis (arrowheads) and were more numerous than in control cells. The asterisk indicates the detached population of keratinocytes. (B) RA-triggered apoptosis in RAR-transduced keratinocytes may be caspase-dependent. The different neoplastic keratinocytes were treated with 3 x 10-6 M RA for 48 h or 72 h and protein extracts from attached and detached populations were prepared using PARP extraction buffer as indicated. The different protein extracts were immunoblotted against PARP and K14 antibodies. The asterisk indicates protein extracts from the detached population of keratinocytes, and the arrow indicates full-length and cleaved PARP. Similar results were observed in two independent experiments.

 
Because epidermal keratinocyte differentiation and cell death are intimately intertwined, squamous differentiation markers were assayed in order to determine whether differentiation accompanies apoptosis in RAR-transduced neoplastic keratinocytes. Frequently expressed in the suprabasal, spinous layers of skin and in differentiated keratinocytes, K1 was not induced in SP-1 RAR{alpha} or RAR{gamma} cells (data not shown). However, PKC{eta} is expressed in differentiated keratinocytes and is up-regulated by 48 h and 72 h post-RA treatment in RAR{alpha} and RAR{gamma} keratinocytes, respectively (Figure 7Go). Interestingly, a simultaneous cleavage product of PKC{eta} appears only in the detached population of cells. Furthermore, PKC{eta} was detected in LXSN-cells but was not up-regulated by RA treatment and cleavage was observed in the detached population of control keratinocytes (results not shown).



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  		Fig. 7. Retinoic acid treatment induces PKC{eta} in RAR-transduced neoplastic keratinocytes. Cells were grown in 100 mm tissue culture plates and treated when 50–60% confluent with 3 x 10-6 M RA or vehicle DMSO for 24, 48, and 72 h. Total SDS protein extracts from attached and detached cells were immunoblotted against PKC{eta} antibody. The arrows indicate the full length PKC{eta} and the observed cleavage product. The asterisk denotes protein extracts from the detached population of keratinocytes. Similar results were observed in three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In these studies, the effect of overexpressed RAR signaling on the growth of neoplastic epidermal keratinocytes was investigated. We found that each epidermal RAR type causes RA induced growth arrest and apoptosis of transformed keratinocytes through distinct as well as overlapping pathways.

Initial attempts to characterize the individual RAR signaling pathways have revealed that RAR isoforms share largely redundant functions. Based upon in vivo knockout animal studies, deletion of a single RAR isoform, such as RAR{alpha}1 or RAR{gamma}2 causes no aberrant phenotype, while RAR{alpha}-null or RAR{gamma}-null animals exhibit similar and/or distinct developmental defects (2,3,41). Interestingly, normal skin morphology remains intact in such RAR{alpha}- or RAR{gamma}- knockout animals (42). These and numerous other observations (41,43) suggest largely overlapping functions for the different RARs, where one receptor type may compensate for the deficiency of another. Since RARs function through heterodimerization with RXRs to act as transcriptional regulators, RXR knockout studies have also helped shed some light on RAR function. Complete disruption of RXR signaling in mice is embryonic lethal, while controlled RXR{alpha} mutations in the epidermis result in abnormal hair cycling and aberrant keratinocyte proliferation and differentiation (44). These results reinforce the need for intact retinoid signaling in the skin but cannot rule out the possible role of other potential RXR heterodimers such as vitamin D receptor in epidermal development and homeostasis.

Only recently, in vivo and in vitro studies have begun to uncover subtle differences in RAR{alpha} and RAR{gamma} functions in the skin. For instance, basal layers of skin overexpressing a dominant-negative RAR fail to differentiate (7). Also, epidermal keratinocytes derived from RAR-knockout animals and harboring a dominant negative p53 protein were recently used to dissect the RAR-signaling pathways during RA-induced growth inhibition (45). In the latter in vitro model, RAR{gamma} signaling provides more potent growth inhibition and repression of AP-1 activity than does RAR{alpha}. However, these studies did not investigate the differential roles of retinoid signaling in the control of keratinocyte cell cycle arrest, apoptosis, and differentiation.

In the present study, a mutant active rasHa oncogene is present in both SP-1 and 308 epidermal cell lines and RAR transcript and proteins were undetectable. We have previously shown that initiation of keratinocytes by activated rasHa and skin tumor progression results in reduced RAR signaling. Here we show that RAR{alpha}-transduced keratinocytes are slightly more sensitive to the RA-induced growth arrest than their RAR{gamma}-transduced counterparts. This discrepancy in RAR{alpha} versus RAR{gamma} sensitivity between our rasHa activated keratinocytes and recently reported p53-mutated keratinocytes (45) may be due to the fact that the former cells are tumorigenic whereas the later ones are not. The possible involvement of p53 signaling in the RA induced growth arrest observed in our RAR-transduced keratinocytes is currently under investigation. In support of our results, RAR{alpha} was found to be the receptor capable of full induction of retinoid responses and G0/G1 cell cycle arrest during retinoid signaling in both estrogen positive and negative human breast cancer cells (23,46).

This observed RAR-induced cell cycle arrest prompted us to investigate the expression patterns of various cell cycle regulators through which each receptor type may exert its functions during G0/G1. The CDKI p21 has been previously reported to play a major role in Ca2+-induced cell cycle arrest and differentiation of primary mouse keratinocytes (47). Our observed RA-induced early, transient up-regulation of p21 protein in both RAR-transduced neoplastic epidermal cells follows a similar time course as does Ca2+-induced up-regulation of p21 in primary mouse keratinocytes (47). In addition to p21 protein up-regulation, RAR{alpha} elicits a similar early, transient up-regulation of the CDKI p16 protein. Interestingly, RAR{gamma}-transduced neoplastic keratinocytes do not show a similar increase in p16 protein levels upon RA treatment. Cyclin D/cdk4,6 complex is a known target for p21 and p16 inhibition (48). However, recent evidence has shown that the Cip/Kip bound cyclin D-dependent kinases remain catalytically active unlike the INK4 proteins that act as inhibitors of these cdks (49).

Cyclin D1 is necessary for progression through G1, and data from experimental and human cancers implicate cyclin D1 as a mediator of ras-induced proliferation (50). It is well established that cyclin D1 is a critical target for oncogenic ras in mouse skin (51). In keratinocytes, the ras oncogene upregulates cyclin D1 and associated kinase expression. In addition, cyclin D1 deficient mice are less susceptible to development of squamous tumors generated through the grafting of activated rasHa-transduced keratinocytes (51). The latter study suggests that the rasHa oncogene signals through cyclin D1 to promote tumorigenesis. Our epidermal cell lines contain a characteristic oncogenic mutation in codon 61 of the rasHa gene and express cyclin D1 proteins. Several studies report an RA-induced decline in cyclin D1 levels that is associated with reduced epithelial tumor development (52). This decrease in cyclin D1 protein occurs through a proteosome-mediated pathway (52). In this study, we show a similar down-regulation of cyclin D1 protein levels that is mostly obvious in RAR{alpha}-transduced keratinocytes. The more pronounced cyclin D1 down-regulation caused by RAR{alpha} may be due to the fact that RAR{alpha}-induced growth arrest is more marked than that observed for RAR{gamma} in these neoplastic keratinocytes. Whether retinoblastoma (Rb) signaling is involved in growth suppression that is induced by the different retinoid receptor pathways has yet to be determined.

Apoptosis seems to be the final fate of the RAR-transduced cells following RA treatment. Interestingly, the observed chromatin condensation and PARP cleavage predominate in the detached apoptotic cell population. In accordance with this, we observed an appreciable amount of cell detachment following RA-treatment of our RAR-transduced cells, while much fewer cells detach upon RA treatment of control neoplastic keratinocytes. This was surprising to us since pharmacological levels of RA inhibit cornified envelop formation in primary epidermal keratinocytes and in normal skin (4,6). The extent of apoptosis and caspase involvement following RA treatment of RAR-transduced neoplastic keratinocytes warrants further investigation.

Because epidermal cell death and differentiation are intimately connected (25), we assayed for epidermal differentiation markers. Calcium-induced epidermal differentiation is associated with up-regulation of numerous squamous markers such as loricrin, filaggrin, and the keratin pair K1/K10 (37). Retinoid treatment of primary epidermal keratinocytes negatively regulates the K1/K10 differentiation markers (6). Consistent with this observation, we were unable to detect any K1 proteins upon RA treatment in the RAR-transduced cells. An alternate marker of keratinocyte differentiation that is not suppressed by retinoid treatment is the novel PKC isoform PKC{eta}. In situ hybridization and immunohistochemical staining show that PKC{eta} is highly expressed in differentiated epithelial cells and is upregulated during later stages of epidermal differentiation (53). Furthermore, overexpression of PKC{eta} in primary keratinocytes inhibits their growth and induces squamous differentiation (54). In accordance with the latter observations, we show an up-regulation of PKC{eta} in both RAR-transduced cell lines upon RA treatment that is simultaneous to the appearance of PARP cleavage. The increase in PKC{eta} was observed earlier in RAR{alpha}-transduced keratinocytes, again indicating a more efficient differentiation program induced by RAR{alpha}. A distinct cleavage product of PKC{eta} at about 50 KD migration appears upon RA treatment in the detached population of keratinocytes. This PKC{eta} cleavage product may indicate a catalytic subunit similar to that observed for PKC{delta} (55). The latter observation is supported by a recent study where an active truncated form of PKC{eta} that is caspase-3 mediated was found in apoptotic lymphocytes (56). Not surprisingly, both PARP cleavage and apparent PKC{eta} cleavage appear in the detached, differentiated RAR-transduced keratinocytes.

Retinoids are widely used for the treatment of numerous malignancies, however, retinoids cause many adverse side effects due to their broad spectrum of action. Retinoid signaling pathways may still be intact early in the neoplastic process and retinoids have been shown to restore RAR signaling in human squamous cell carcinoma cells (57). Therefore, a better understanding of the regulation of cell proliferation and death by retinoids is beneficial to help fine-tune a more targeted approach to their clinical use. A combination of retinoid receptor gene transfer and the administration of retinoid receptor-specific agonists may be a more efficient strategy for skin cancer treatment. Alternatively, in the absence of RARs, synthetic retinoids that function through retinoid receptor-independent pathways may be used for prevention or therapy of skin cancer.


   Notes
 
5 To whom correspondence should be addressed at: Biology Department, American University of Beirut, PO Box 11-0236, Beirut, Lebanon Email: darwichn{at}aub.edu.lb Back


   Acknowledgments
 
This work was supported by grants from the American University of Beirut University Research Board, the Lebanese National Council for Scientific Research, Medical Practice Plan, Diana-Tamari Sabbagh Funds, and Terry Fox Cancer Research Funds.

We are especially grateful to Dr Steven S.Collins for providing the retroviral packaging cell lines and Dr Fadia Homeidan for her critical review of the manuscript.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 21, 2001; revised August 9, 2001; accepted August 27, 2001.


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