OVCA2 is downregulated and degraded during retinoid-induced apoptosis

Authors


Abstract

Retinoids, the natural and synthetic derivatives of vitamin A, have been shown to regulate the growth and differentiation of a wide variety of cell types and consequently have enormous potential as chemotherapeutic agents. We have previously identified 2 genes, termed OVCA1 and OVCA2, which are located in a small region showing a high frequency of allelic loss in breast and ovarian tumors and share a common exon. Recent studies have suggested that expression of OVCA1 may be influenced by retinoids. Therefore, we analyzed the expression of OVCA1 and OVCA2 in cells in response to treatment with all-trans retinoic acid (RA) and N-(4-hydroxyphenyl)retinamide (4HPR), or under conditions of low serum and confluence, to determine further the roles of OVCA1 and OVCA2 in cell growth, apoptosis and differentiation. We show that OVCA2 mRNA and protein are ubiquitously expressed and that they are downregulated in the lung cancer cell line Calu-6 after treatment with RA and 4HPR. In addition, we observed that OVCA2 protein is proteolytically degraded in response to RA and 4HPR treatment in a time- and dose-dependent manner in the promyelocytic leukemia cell line HL60. In contrast, expression of the candidate tumor suppressor OVCA1 was not downregulated by these treatments. Furthermore, we demonstrate that OVCA2 is evolutionarily conserved and shows regional homology with dihydrofolate reductases (DHFRs), specifically with hydrolase folds found in α-β hydrolases. Our results are in contrast to a previous report and show that OVCA2, not OVCA1 mRNA and protein, is downregulated in response to RA and 4HPR. © 2002 Wiley-Liss, Inc.

Retinoids, the natural and synthetic derivatives of vitamin A, have been shown to regulate the growth and differentiation of a wide variety of cell types and consequently have enormous potential as chemotherapeutic agents.1, 2 The diverse effects of retinoids are mediated by binding to at least 6 retinoid receptors, which fall into 2 subfamilies: retinoic acid receptors (RARs) α, β and γ and the retinoid X receptors (RXRs) α, β and γ.3 The RARs and RXRs act as transcription factors, binding as homo- and heterodimers to retinoid response elements in the promoter regions of target genes and thus enhancing or repressing transcription. In addition, RARs and RXRs can inhibit the expression of AP1-dependent genes by antagonizing AP1 activity.4–6 However, many of the downstream targets that lead to retinoid-induced growth arrest, differentiation and/or apoptosis remain to be identified. In addition, synthetic retinoids, such as 4HPR, which have been developed as chemoprevention agents with an acceptable toxicity profile, may well differ in their mechanism of action.7–9

Loss of heterozygosity (LOH) at 17p13.3 has been reported in ovarian tumors, breast tumors, primitive neuroectodermal tumors, carcinoma of the cervix uteri, medulloblastomas and lung tumors, suggesting that genes on 17p13.3 may play a role in the development of multiple cancers.10–18 We and others have previously defined a minimum region of allelic loss (MRAL) on chromosome 17p13.3 in genomic DNA from ovarian tumors.19, 20 Positional cloning and sequencing techniques revealed at least 2 candidate tumor suppressor genes in the ∼20 kb MRAL, referred to as OVCA1 or DPH2L and OVCA2.19, 20 The OVCA1/DPH2L and OVCA2 genes overlap one another in the MRAL and have 1 exon in common.20 Since translation of OVCA1/DPH2L does not proceed into the shared exon (exon 13 in OVCA1/DPH2L and exon 2 in OVCA2), the genes encode for completely distinct OVCA1 and OVCA2 proteins.

We have previously shown that OVCA1 is a strong candidate for a tumor suppressor gene: it is downregulated in a proportion of breast and ovarian tumors, and overexpression of OVCA1 reproducibly inhibits colony formation in a variety of tumor cell lines.21 However, recent studies have suggested that OVCA1 may be downregulated after differentiation or growth arrest induced by RA in lung cancer cell lines, contrary to a role as a tumor suppressor.22 Therefore, we have analyzed both OVCA1 and OVCA2 mRNA and protein expression in cell lines treated with all-trans retinoic acid (RA) and N-(4-hydroxyphenyl)retinamide (4HPR), or under conditions of low serum and confluence, to determine a possible role of OVCA1 and/or OVCA2 in cell growth, apoptosis and differentiation. We show that OVCA2 mRNA and protein is downregulated in the lung cancer cell line Calu-6 treated with RA and 4HPR, but that OVCA1 is unaffected. Interestingly, OVCA2 protein is degraded in response to RA and 4HPR treatment in the promyelocytic leukemia cell line HL60. In addition, we present a further characterization of OVCA2 and show that it is a highly conserved gene that is related to a variety of α-β hydrolases including esterases, lipases and other enzymes.

MATERIAL AND METHODS

Cell lines

Cos-1, MCF-7, SKOV-3, HeLa and A2780 cells were maintained in DMEM supplemented with 10% FCS, glutamine and insulin. A549 cells were maintained in Kaighn's modification of Ham's F12 medium supplemented with 10% FCS and glutamine. HL-60 cells and Calu-6 cells were maintained in RPMI-1640 supplemented with 10% FCS and glutamine. Human ovarian surface epithelial cell lines expressing SV-40 large T-antigen (HIO cells) have been previously described.23

Cell culture treatments

RA (Sigma, St. Louis, MO) and 4HPR (NIH) were dissolved in ethanol at a stock concentration of 10 mM. Calu-6 and A549 cells were seeded at equal density (5 × 105 cells) in 100 mm dishes and allowed to attach for 24 hr. They were then treated with either 10 and 1 μM RA or 4HPR or ethanol alone for 24, 48, 72 or 96 hr. HL60 cells, which are nonadherent, were seeded into T25 flasks at equal density (1 × 105 cells) and treated with 1 or 0.1 μM RA and 4HPR or ethanol alone for 24, 48, 72 and 96 hr. A549 cells were cultured in 1% FCS for 1 week or cultured for 3 days after confluence.

Preparation of RNA and protein extracts

Total cellular RNA was isolated using guanidinium/isothiocyanate/phenol/chloroform as previously described.20 Whole cell protein extracts were made by incubating cells in PBSTDS (10 mM Na2HPO4, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% SDS, 0.2% NaN3, 1 mM EDTA, 5 mM NaF, 100 μg/ml PMSF, 1 μg/ml leupeptin, 0.7 μg/ml pepstatin, pH 7.25) as described previously.21 Quantitation of protein was determined using a bicinchoninic acid/copper (II) sulfate assay (Sigma). Extracts from normal human tissues were purchased from ClonTech (Palo Alto, CA).

Northern blot analysis

Fifteen micrograms of total RNA was used for standard Northern blot analysis, as previously described.20 Multiple tissue Northern blots containing 5 μg of poly(A)+-selected mRNA from various human tissues were purchased from Clontech. Blots were hybridized with a ∼830 bp cDNA probe corresponding to exon 13 of OVCA1 and exon 2 of OVCA2 or a ∼200 bp cDNA probe corresponding to exon 1 of OVCA2.

Antibodies

Anti-β-actin was purchased from Sigma. Anti-PARP antibody was purchased from Cell Signaling (Beverly, MA). The anti-OVCA1 antibody TJ132 has been described previously.21 For the production of anti-OVCA2 antibodies, a peptide corresponding to amino acids 27–41 of OVCA2 was synthesized (Research Genetics, Huntsville, AL). Purity of the peptide was confirmed by high-performance liquid chromatography. The peptide was conjugated to malemide-activated keyhole limpet hemocyanin (Pierce, Rockford, IL) and used to immunize a New Zealand White rabbit (Cocalico Biologicals, Reamstown, PA). Two milligrams of antigenic peptide were covalently linked to Aminolink agarose (Pierce) and used to purify anti-OVCA2 antibody according to the manufacturer's instructions. The final antibody was referred to as TJ143.

Caspase-3 activity assay

Colorimetric CaspACE assay (Promega, Madison, WI) was used to detect caspase-3 activity. HL60 cells treated with 1 or 0.1 μM RA or 4HPR for 24, 48, 72 and 96 hr or with ethanol alone were harvested, snap-frozen and stored at −70°C. The frozen cell pellet was lysed (20 μl lysis buffer per million cells) by 1 cycle of freezing and thawing. Twenty micrograms of cell lysate (20–50 μg total protein) was incubated with the caspase-3 substrate Ac-DEVD-pNA for 4 hr at 37°C. Cleavage of the substrate to release pNA was monitored at 405 nm using a microplate.

FACS analysis

HL60 cells treated with 1 μM RA or 4HPR or ethanol alone for 96 hr were harvested and stained with propidium iodide. Fluorescent cells were analyzed using a FACScan machine running CellQuest software (Becton Dickinson, Franklin Lakes, NJ). The percentage of cells in each stage of the cell cycle was calculated by the Watson Pragmatic algorithm, using FlowJo software (Tree Star, San Carlos, CA).

cDNA cloning and cell transfections

Cloning of the full-length OVCA2 cDNA and genomic DNA was previously described.20 Genomic OVCA2 DNA was subcloned into the mammalian expression vector pcDNA3 (InVitrogen, La Jolla, CA) by PCR-amplifying a DNA fragment using gene-specific primers containing BamHI (5′) or EcoRI (3′) restriction endonuclease sites, digesting the fragment and cloning it into the multiple cloning sequence of pcDNA3. To produce an N-terminal hemagglutinin (HA)-tagged OVCA2 expression vector, OVCA2 cDNA was first cloned into the HA-containing mammalian expression vector J3H, and then the HA-OVCA2 cDNA was subcloned into pcDNA3. Cell lines were transfected during the log phase of growth with 5 μg of vector using the Superfect reagent (Qiagen, Chatsworth, CA), according to the manufacturer's instructions.

SDS-PAGE and western blot analysis

Fifty micrograms of total protein extract from tissues or 30 μg of total protein from cell extracts, unless otherwise stated, were separated by standard SDS-PAGE and transferred to Immobilon-P (Millipore, Bedford, MA). The membranes were blocked either in 3% BSA and probed with anti-OVCA1 antibody TJ132 or in 5% dried milk and probed with the anti-OVCA2 antibody TJ143 or the anti-β-actin antibody (Sigma).

Sequence analysis

GenBank/EMBL and SwissProt sequences showing homology to OVCA2 were identified using the basic local alignment search tool (BLAST, NCBI). DNA and amino acid sequence comparisons and motif analyses were performed with the Wisconsin Package versions 8 and 9.1 (Genetics Computer Group).

Homology modeling of OVCA2

We used methods described previously to build a model of OVCA2.24, 25 Briefly, PSI-BLAST was used to build a sequence profile of OVCA2 by iteratively searching the nonredundant protein sequence database available from the NCBI.26 Only sequences with expectation values better than 0.0001 were included in the sequence profile matrix. Upon completion, this matrix was used to search a database of protein sequences in the Protein Data Bank27 of experimentally determined protein structures. A model of OVCA2 was built using the side-chain conformation prediction program SCRWL,28 which works by building side chains on a template backbone by first placing residues according to a backbone-dependent rotamer library,29 followed by a combinatorial search to remove steric overlaps.

RESULTS

Analysis of OVCA1 and OVCA2 under conditions of growth arrest and apoptosis

We have previously reported the isolation of 2 candidate tumor suppressor genes, referred to as OVCA1 and OVCA2.20 A schematic of the 2 genes is shown in Figure 1. The full cDNA sequence of OVCA1 and OVCA2 has been deposited into GenBank (accession numbers AF335321 and AF321875, respectively). Recent studies suggested that OVCA1 was downregulated in response to cell differentiation, growth arrest and apoptosis induced by RA and 4HPR in the lung cancer cell lines Calu-6 and GLC-82 and the promyelocytic leukemia cell line HL60.22 Since the results were in sharp contrast to our previous findings of dramatic growth suppression induced by overexpression of OVCA1, we elucidated the expression of OVCA1 and OVCA2 in response to RA and 4HPR. Initially we analyzed the lung cancer cells Calu-6 and A549, using the same treatments described by Liu and colleagues,22i.e., cells were treated with 10 μM RA and 4HPR for a period of 4 days.

Figure 1.

Genomic organization of OVCA1 (exons shown in black boxes) and OVCA2 (exons shown in white boxes). OVCA1 and OVCA2 map to a 20 kb minimum region of allelic loss in ovarian tumors, between the markers D17S28 and D17S5/30, at 17p13.3.20 The OVCA1 and OVCA2 genes overlap one another and have 1 exon in common (exon 13 of OVCA1 and exon 2 of OVCA2). Since translation of OVCA1 does not proceed into exon 13 in OVCA1 or exon 2 in OVCA2, the genes encode for completely distinct OVCA1 and OVCA2 proteins. Probes used for Northern hybridizations are shown.

To determine whether the RA and 4HPR treatments were affecting the growth properties of our cell lines, we performed direct cell counts (Fig. 2a). Both cell lines treated with RA and 4HPR exhibited a decrease in cell number compared with control (Fig. 2a). We analyzed the expression of OVCA1 and OVCA2 after RA and 4HPR treatment by Northern blot analysis using a probe to exon 2/13 of OVCA1/2 (Fig. 1). As shown in Fig. 2b, ≃2.4 and 1.3 kb transcripts were expressed in both cell lines, corresponding to OVCA1 and OVCA2 respectively.20 The 1.3 kb OVCA2 transcript was downregulated in both Calu-6 and A549 cells in response to RA and 4HPR treatment. Liu et al.22 also found that a ∼1.7 kb transcript was reproducibly downregulated in Calu-6 cells after a 4-day treatment with RA or 4HPR; however, the authors interpreted the ∼1.7 kb transcript to be a smaller transcript of OVCA1/DPH2L. The 2.3 kb OVCA1 transcript was unaffected by RA treatment in both cell lines. We did observe slight variations (<1-fold) in OVCA1 mRNA levels in A549 and Calu-6 cells treated with 4HPR; however, β-actin normalized levels were unchanged (Fig. 2b).

Figure 2.

(a) Growth inhibition of cells treated with all-trans retinoic acid (RA) and N-(4-hydroxyphenyl)retinamide (4HPR).Cells treated with 10 μM RA or 4HPR for 4 days were harvested and stained for trypan blue uptake, and unstained cells were counted using a hemocytometer. The mean relative cell number as a percentage of the control is shown for both cell lines from 3 separate experiments, together with the standard deviation, shown as error bars. (b) Analysis of OVCA2 and OVCA1 mRNA expression in Calu-6 and A549 cells treated with 10 μM RA or 4HPR for 4 days compared with control cells (ethanol alone). Fifteen micrograms of total RNA from the indicated cell lines and treatments were used for standard Northern blot analysis. The Northern blot was hybridized with a probe corresponding to exon 13 of OVCA1 and exon 2 of OVCA2, or with a probe to β-actin, as a control, as indicated. Lower panel: Ethidium bromide-stained gel prior to blotting. The position of the 28S and 18S rRNA is indicated. (c) Analysis of OVCA2 and OVCA1 protein expression in Calu-6 and A549 cells treated with 10 μM RA and 4HPR compared with control cells (ethanol alone) for 4 days. Thirty micrograms of extracts from the indicated cell lines and treatments were separated by 12% SDS-PAGE and processed by Western blotting. The blots were probed with the anti-OVCA2 antibody TJ143 or the anti-OVCA1 antibody TJ132. Anti-β-actin was used a loading control. A representative figure is shown from 2 separate experiments. (d) Analysis of the effects of confluence and low serum on OVCA2 and OVCA1 expression. A549 cells were growth-arrested by culturing in 1% serum for 1 week or for 3 days after confluence. Thirty micrograms of extracts from the indicated treatments were separated by 12% SDS-PAGE and processed by Western blotting. The blots were probed with the anti-OVCA2 antibody TJ143 or the anti-OVCA1 antibody TJ132. Anti-β-actin was used a loading control. A representative figure is shown from 2 separate experiments.

To clarify further the effect of RA and 4HPR on OVCA1 and OVCA2, we analyzed their protein expression after 4-day treatment with 10 μM RA or 4HPR by Western blot analysis. Antibodies against amino acids 27–41 of OVCA2 were generated by injecting the peptide into rabbits, and the antiserum was immunoaffinity purified (referred to as TJ143). The anti-OVCA1 antibodies TJ132 have been described previously.21 In Calu-6 cells OVCA2 protein levels were downregulated by RA and 4HPR, but OVCA1 was not significantly affected (Fig. 2c). In A549 cells, neither OVCA2 nor OVCA1 protein levels were measurably affected by RA or 4HPR treatment (Fig. 2c). Furthermore, OVCA2 protein levels were unaffected by growth arrest induced by cell confluence for 3 days or low serum for 1 week in A549 cells, whereas OVCA1 levels were decreased >2-fold in serum-deprived cells (Fig. 2d).

The effect of RA and 4HPR on OVCA1 and OVCA2 protein levels in Calu-6 and A549 cells was further evaluated. Cells were treated with 1 or 10 μM RA or 4HPR for 24, 48, 72 or 96 hr. In Calu-6 cells, OVCA2 protein only appeared to be downregulated after 96 hr of treatment with 10 μM RA and after 48 hr of treatment with 10 μM 4HPR (Fig. 3). OVCA1 protein was not affected by these treatments in Calu-6 cells. In A549 cells, OVCA2 was slightly downregulated after 48, 72 and 96 hr of treatment with 10 μM RA and 4HPR. OVCA1 protein levels appeared to increase after 96 hr of treatment with 10 μM 4HPR; however, this increase was not always consistently observed. Neither OVCA1 nor OVCA2 were affected by the lower dose of 1 μM RA and 4HPR during these time points in either cell line (data not shown). Overall, the results suggest that, in contrast to the conclusions from a previous report, OVCA2, not OVCA1 can be downregulated in response to RA and 4HPR treatment.

Figure 3.

Analysis of OVCA2 and OVCA1 protein expression in Calu-6 and A549 cells lines treated with 10 μM all-trans retinoic acid (RA) and N-(4-hydroxyphenyl)retinamide (4HPR) compared with control cells (ethanol alone) for 24, 48, 72 and 96 hr. Thirty micrograms of extracts from the indicated cell lines and treatments were separated by 10% SDS-PAGE and processed by Western blotting. The blots were probed with the anti-OVCA2 antibody TJ143 or the anti-OVCA1 antibody TJ132. Anti-β-actin was used a loading control.

OVCA2 is degraded in HL60 cells in response to RA and 4HPR treatment

We also analyzed the effect of RA and 4HPR on OVCA2 expression in HL60 cells. Previous studies have shown that in HL-60 cells RA promotes differentiation, followed by apoptosis, and that 4HPR induces apoptosis.30, 31 Cells were treated with 0.1 and 1 μM RA and 4HPR, and the mRNA and protein levels were evaluated by Northern and Western blotting, respectively. As expected, the mRNA levels were reduced >2-fold (data not shown). Interestingly, the OVCA2 protein levels were dramatically reduced in response to RA and 4HPR treatments (Fig. 4a). The level of 25 kDa full-length protein appeared to correlate directly with the length of treatment and the drug dosage of both RA and 4HPR (Fig. 4a). HL60 cells are nonadherent, and therefore this may be an indication of the number of dead cells, which, in contrast to attached cell lines such as Calu-6 and A549, are not removed during refeeding and when harvested. Thus, the protein degradation may be a result of RA- and 4HPR-induced apoptosis. To determine whether the HL60 cells were indeed undergoing apoptosis, we performed FACS analysis and analyzed caspase 3 activity. We did not see an increase in caspase 3 activity as assessed using a Colorimetric CaspACE assay and PARP cleavage (data not shown). However, the FACS analysis did show an increase in the sub G1/G0 fraction, indicating increased cell death, as well as a dramatic arrest in G1/G0 (Fig. 4b). These results suggest that OVCA2 may be targeted for protein degradation after RA- or 4HPR-induced differentiation and/or apoptosis.

Figure 4.

(a) Analysis of OVCA2 protein expression in HL60 cells treated with 0.1 μM and 1 μM all-trans retinoic acid (RA) and N-(4-hydroxyphenyl)retinamide (4HPR) compared with control cells (ethanol alone) for 24, 48, 72 and 96 hr. Fifty micrograms of extracts from the indicated treatments were separated by 10% SDS-PAGE and processed by Western blotting. The blots were probed with the anti-OVCA2 antibody TJ143. (b) FACS analysis of HL60 cells treated with 1 μM RA or 4HPR or ethanol alone for 96 hr. Treated cells were harvested and stained with propidium iodide and analyzed by FACS. The percentage of cells in each stage of the cell cycle is indicated.

OVCA2 has a broad tissue distribution

To characterize OVCA2 further, we analyzed OVCA2 mRNA expression in a variety of tissues. Multiple tissue Northern blots were probed with the exon 2/13 of OVCA1/2 or the unique exon 1 of OVCA2, as depicted in Figure 1. As shown in Figure 5a, all tissues exhibited 2 bands (∼2.4 and 1.3 kb). When our blots were reprobed with an exon 1 probe of OVCA2, all tissues tested exhibited only the 1.3 kb band representing the OVCA2 transcript, with testis, heart, skeletal muscle, liver and pancreas showing high mRNA expression (Fig. 5b).

Figure 5.

Tissue expression pattern of OVCA1 and OVCA2 mRNA. Blots containing 5 μg of polyA+ selected mRNA from each of the indicated human tissues were hybridized with a ∼830 bp cDNA probe corresponding to exon 13 of OVCA1 and exon 2 of OVCA2(a) or a ∼200 bp cDNA probe corresponding to exon 1 of OVCA2(b). Size standards are in kilobases.

In addition, we analyzed OVCA2 protein expression in a number of cell lines and tissues. Western blot analysis using the anti-OVCA2 antibody TJ143 revealed that Cos-1 cells, transfected with a genomic DNA fragment containing the 2 exons of OVCA2 under the control of a CMV promoter, produced the predicted ∼25 kDa protein (Fig. 6a). The same results were obtained when Cos-1 cells were transfected with the OVCA2 cDNA (not shown), indicating that the mRNA transcribed from the genomic DNA was correctly spliced within the cells. The antibody also detected endogenous OVCA2 in various breast and ovarian cell lines (Fig. 6a) and in a variety of human tissues (Fig. 6b). Of the tissues analyzed, the kidney, liver, testis, placenta and thymus all exhibited high levels of OVCA2 (Fig. 6b).

Figure 6.

(a) Characterization of OVCA2 expression. Twenty micrograms of extracts from the indicated cell lines were separated by 12% SDS-PAGE and processed by Western blotting. The blot was probed with the anti-OVCA2 antibody, TJ143. Lane Cos-1/OVCA2, extract of Cos-1 cells that had been transfected with pcDNA3-OVCA2; lanes HIO-118, HIO-135, HIO-117, extracts from SV40 Tag-immortalized human ovarian surface epithelial cell lines (HIO); lane primary ovarian cell line, extract from human ovarian surface epithelial cell line; lanes A2780 and SKOV3, extracts from ovarian cancer cell lines; lane MCF-7, extract from breast cancer cell line. (b) OVCA2 expression in human tissues. Fifty micrograms of extracts from the indicated human tissues (Clontech) were separated by 12% SDS-PAGE and processed by Western blotting. The blot was probed with the anti-OVCA2 antibody TJ143.

OVCA2 is highly evolutionarily conserved

The OVCA2 protein consists of 227 amino acids (Fig. 7). A BLAST search of GenBank/EMBL and SwissProt databases revealed that OVCA2 does not match any known mammalian genes. However, 1 C. elegans and 4 yeast proteins were identified that exhibited up to 60% similarity and up to 45% identity to the amino acid sequence of OVCA2 and that contained a similar number of amino acids (Fig. 7). These sequences were described as putative dihydrofolate reductases (DHFRs), but they share more conserved domains with OVCA2 than with mammalian DHFRs (data not shown). A BLAST search of the EST database revealed full-length mouse and partial rat OVCA2 homologs displaying 87% and 86% similarity, respectively, to the amino acid sequence of OVCA2. In addition, 2 plant ESTs (rice and arabidopsis; up to 53% similar) and multiple human sequences were identified. A multiple sequence alignment of OVCA2 with all available non-human OVCA2 homologs (Fig. 7) revealed at least 5 conserved domains, which presently have no known function, but which may be important new functional domains based on their evolutionary conservation. Zoo blots probed with the unique exon 1 of OVCA2 demonstrated that all mammalian species tested have an OVCA2 homolog (data not shown). Interestingly, when exon 2 of OVCA2, which is a noncoding exon of OVCA1, was used to probe these blots, both OVCA2 and OVCA1 bands were identified, suggesting that the genomic arrangement of the 2 genes is conserved among many different species. This high degree of evolutionary conservation suggests that OVCA2 may be very important for normal cellular function.

Figure 7.

Multiple sequence alignment of OVCA2 amino acid sequence and similar sequences from mouse, rat, C. elegans, S. cerevisiae, S. pombe, rice and arabidopsis. At least 5 conserved regions are evident. Two protein kinase C phosphorylation sites (PKC), 2 casein kinase-2 sites (CK2), a potential pseudo-leucine zipper motif and a potential MYB DNA binding site (MYB-DNA) are all indicated.

The Genetics Computer Group package was used to evaluate functional motifs within the OVCA2 amino acid sequence (Fig. 7). Two protein kinase C phosphorylation sites (amino acids 18 and 178), 2 casein kinase-2 phosphorylation sites (amino acids 76 and 84) and a possible leucine zipper variant (amino acid 95) were identified, all of which are conserved within the available mouse and rat sequences. In addition, a MYB DNA binding motif was observed (amino acid 83), which is identical to the native MYB motif, except for a conservative amino acid change from tryptophan to phenylalanine. Interestingly, this domain contains 1 of the casein kinase-2 phosphorylation sites. No other functional groups were identified that could provide clues to the function of OVCA2.

OVCA2 protein model

Sequence analysis using PSI-BLAST indicated that OVCA2 is related to the N-terminal domain of some DHFRs, notably DYR_SCHPO, a DHFR in yeast. This domain has a hydrolase fold that is found in α-β hydrolases including esterases and lipases and includes the 3 residues of the catalytic triad, Asp, Ser, His (Fig. 8). The crystal structure of Protein Data Bank entry 1AUR32 was used to build a model of OVCA2 using the side chain conformation prediction program SCWRL.28 The sequence identity between OVCA2 and the crystal structure is only 13%, but the same fold was identified with high confidence with 3 different programs, PSI-BLAST,26, 3d-pssm33 and Threader.34 The significance of this domain within the DHFRs, however, has yet to be reported.

Figure 8.

Molecular model of OVCA2 shown as a ribbon diagram. The 3 residues of the catalytic triad conserved in α-β hydrolases are shown as stick figures. The model was built from Protein Data Bank entry 1AUR.31

DISCUSSION

We have found that expression of OVCA2 but not OVCA1, is downregulated in cells in response to RA and 4HPR. This is in contrast to a recent paper by Liu et al.,22 in which it was reported that OVCA1/DPH2L mRNA levels were decreased in several cancer cell lines after treatment with RA or 4HPR. The authors used mRNA differential display to uncover genes modulated by RA in human lung cancer cell lines, and a clone was identified that was homologous to the 3′UTR of OVCA1/DPH2L. They performed Northern blot analysis with a probe to a 3′ fragment of OVCA1/DPH2L that detected both a ∼2.3 kb trancript and a ∼1.7 kb transcript, which is consistent with our Northern blot data when probed with exon 2/13 of OVCA1/2. We20 and Phillips et al.35 have previously described the 2.4 kb/2.3 kb transcript as OVCA1/DPH2L. However, Liu and colleagues22 interpreted the ∼1.7 kb transcript to be a smaller transcript of OVCA1/DPH2L. We have now clarified that the 1.7 kb/1.3 kb transcript encodes for OVCA2, which is an entirely different protein from OVCA1/DPH2L.

We have found that the OVCA2 mRNA is downregulated in response to 10 μM RA and 4HPR in the lung cancer cell lines A549 and Calu-6. Liu and colleagues22 also found that the 1.7 kb transcript, which we have now shown corresponds to OVCA2, is downregulated in Calu-6 cells and slightly downregulated in A549 cells. We have evaluated the sequence of the OVCA2 promoter for potential retinoic acid response elements (RAREs) and AP1 binding sites. Our initial screen of the first 10 kbp of genomic sequence upstream of exon 1 failed to uncover any of the common RARE consensus sequences.36 However, we did identify 3 putative AP1 binding sites, and it is possible that OVCA2 may be downregulated because retinoid receptors antagonize AP1 activity.6

At the protein level, which is a more relevant biologic marker for cellular processes, the effect on OVCA2 in A549 cells appears to be less dramatic, and therefore there must be some compensation in OVCA2 expression at the posttranscriptional level. Indeed, there are also differences in mRNA and protein levels seen in the same tissue type from the multiple tissue Northern and Western blots, although the tissue sources are different and therefore it is difficult to do a direct comparison. In Calu-6 cells, OVCA2 protein was downregulated after RA and 4HPR treatment; however, this only occurred with high doses (10 μM), and after 3–4 days. The most dramatic and interesting effect was seen with HL60 cells in which OVCA2 was degraded in response to RA- and 4HPR-induced cell differentiation and apoptosis. It has been shown that HL60 cells undergo apoptosis in response to RA and 4HPR,30, 31 and our FACS data are consistent with increased cell death; therefore this may be a consequence of apoptosis. However, we did not find any consensus cleavage sites for caspases 1, 3, 6, 8 and 9 within the OVCA2 sequence, and we did not detect any increase in caspase 3 activity in response to the treatments; therefore it is unlikely that OVCA2 is a substrate for the caspases.

Further investigations are required to determine how OVCA2 is being proteolytically cleaved, how this is related to RA and 4HPR treatment and the functional consequence of this downregulation. In Calu-6 cells, which have previously been shown to undergo apoptosis in response to RA and 4HPR,22, 37 it is still not clear whether the decrease seen in OVCA2 protein levels is due to transcriptional, posttranscriptional and/or posttranslational processes. Although we did not see the degraded product in Calu-6 cells, this may be because the dead cells are removed during feeding and before harvesting.

In contrast to OVCA2, OVCA1 is not significantly affected in response to 10 μM RA or 4HPR in Calu-6 and, if anything, is slightly upregulated in response to 4HPR in A549 cells. We have shown that overexpression of OVCA1 reproducibly inhibits colony formation in several ovarian tumor cell lines and that stable expression of exogenous OVCA1 expression is difficult to obtain, which is consistent with but is not proof of a tumor suppressor function.21 However, overexpression of OVCA2 in a variety of tumor cell lines has no obvious effects on growth (Prowse and Godwin, unpublished data). The fact that OVCA2 is downregulated in Calu-6 and HL-60 cells also suggests that OVCA2 is not likely to be a tumor suppressor.

The MRAL, which we have mapped in ovarian tumors,20 is in fact only 20 kb, and our mapping studies indicate that there are only 3 genes in this region, OVCA1 and OVCA2, which we have previously reported,20 and OVCA4, which is a testis-specific gene (Godwin, unpublished data). It therefore seems likely that OVCA1, not OVCA2, is the likely tumor suppressor gene in this region. It is of interest that the Calu-6 and A549 cell lines analyzed by Liu et al.22 exhibited no OVCA1 transcript by Northern blot analysis, but our analyses do show OVCA1 mRNA and protein in these cell lines. This could be due to analyzing different subpopulations of the cell lines. Their cell lines are of interest since the lack of OVCA1 transcript suggests that OVCA1 could be a tumor suppressor gene involved in the development of lung tumors. Indeed, studies have shown that LOH at 17p is one of the most frequent alterations in lung cancer.15, 16, 38 In addition, LOH at 17p13.3 is more frequent than at 17p13.1, where TP53 maps, and it appeared to occur in the absence of TP53 mutation and/or 17p13.1 deletion.15, 16, 38 It will be important to investigate further the role of OVCA1 in the development of lung cancer. In addition, it will be of interest to analyze the effect that RA and 4HPR have on OVCA1 expression in breast and ovarian cell lines.

In summary, OVCA2 is a novel gene identified on chromosome 17p13.3. OVCA2 is composed of 2 exons: a unique exon 1 and an exon 2, which comprise part of the 3′ untranslated region of OVCA1. Thus, the 2 genes are overlapping, but their protein products are completely distinct. Both OVCA1 and OVCA2 are highly conserved, suggesting they have important roles in the cell. The homology of OVCA2 to α-β hydrolases suggests that it may have some enzymatic activity; however, further studies are required to determine the significance of this observation. Further analysis of the function(s) of OVCA2 will help to determine the role of OVCA2 role in retinoid-induced growth arrest, differentiation and apoptosis.

Acknowledgements

A.K.G. was the recipient of National Institutes of Health grant RO1 CA-70328, United States Army Medical Research grant DAMD17-96-1-6088 and grants from the Ovarian Cancer Research Fund and the Eileen Stein Jacoby Fund. R.L.D. was the recipient of a grant from the American Cancer Society.

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