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Departments of Obstetrics and Gynecology (O.K., G.H.), Internal Medicine (T.G.), Pathology (L.W.M.), and Urology (T.R.M.), Harbor-UCLA Medical Center, Torrance, California 90502; Department of Obstetrics and Gynecology (M.G., T.G.G.), University of Wisconsin Medical School and Wisconsin National Primate Research Center, Madison, Wisconsin 53792; Department of Biochemistry and Pharmacy (M.S.J., K.A.D.), Åbo Akademi University, Turku, Finland; and Department of Clinical Chemistry (B.W.), VU University Medical Center, Amsterdam, The Netherlands
Address all correspondence and requests for reprints to: Omid Khorram, M.D., Ph.D., Department of Obstetrics and Gynecology, Box 489, Harbor-UCLA Medical Center, 1000 West Carson Street, Torrance, California 90502. E-mail: okhorram{at}obgyn.humc.edu.
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Introduction |
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Materials and Methods |
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Reverse transcription with 1 µg total RNA from human endometrial biopsies spanning d 16–28 of the menstrual cycle (provided by B. Lessey, University of North Carolina-Chapel Hill) was performed using GeneAmp RNA PCR core kit (Perkin-Elmer, Foster City, CA) with Oligo d (T) 16 primers according to instructions. The PCR and primer sequences used were based on a previous report (1). Briefly PCR was carried out according to protocol published elsewhere (1) using 12.5 pmol of each putative inducible nitric oxide synthase (iNOS) primer (forward primer: 5'-CCATTGAAAGTCTTGGTC-3' and reverse primer: 5'-ACTTATCCATGCAGACAAC-3'), 2.5 mM of each deoxynucleotide triphosphate, 50 mM KCl, 10 mM Tris-HCL (pH 8.3), 1.5 mM MgCl2, 0.01% (wt/vol) gelatin, and 5 µl of cDNA template in a final volume of 50 µl. Reactions were amplified as follows: 94 C for 30 sec, 46 C for 30 sec, and 72 C for 45 sec for 35 cycles. Reaction products were run on 2.5% agarose gels and visualized by SYBR Gold (Molecular Probes, Eugene, OR) and analyzed with a Storm 840 PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Semiquantitative analysis of mRNA expression was accomplished by densitometry of the captured data with ImageQuant software (Molecular Dynamics). The amplified reaction products termed MG 30.4 were cloned into the pCR 2.1 TA cloning vector for sequencing (Invitrogen, Carlsbad, CA). 5'-rapid amplification of cDNA ends (RACE) and 3'-RACE were also performed with endometrial or JEG cell RNA. JEG cells cultured in DMEM with 10% fetal calf serum are human choriocarcinoma cells and are a commonly used trophoblast-derived cell line. 5'-RACE was performed using three antisense primers based on the 5' end of the coding region. 3'-RACE was performed using a sense primer (Table 1
) mapping to the 3' end of MG30.4. RACE products were cloned into pCR2.1 for sequencing.
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Tissues specimens were obtained from women undergoing gynecologic surgery for benign and malignant conditions. Two groups of postmenopausal women were studied. Group 1 consisted of women not taking HRT, with a mean age of 60 yr (56–73 yr), and group 2 consisted of women taking daily continuous HRT with a mean age of 66 yr (52–74 yr). These women were undergoing hysterectomy for benign gynecologic reasons including prolapse, menometrorrhagia, and uterine myomas. We also obtained endometrial cancer tissues from postmenopausal women for immunohistochemical studies. For the menstrual cycle study, endometrial biopsies (kindly provided by Dr. Bruce Lessey, University of North Carolina-Chapel Hill) were obtained using a Pipelle curet. Biopsies were timed based on the LH surge and dated on the basis of histologic evaluation (2). These patients were fertile (36–40 yr old), regularly menstruating women who were undergoing tubal ligation and gave written consent for use of the tissue for research. Institutional review board approval was obtained at the University of Wisconsin, Madison; Harbor-UCLA Medical Center, Torrance, CA; and University of North Carolina, Chapel Hill. Tissues for mRNA analysis were frozen and stored at –70 C.
In situ hybridization
Tissues removed during surgery were immediately immersed in Bouin’s fixative, embedded in paraffin, and cut into 5-µm-thick sections. For probe generation, a 259-bp fragment corresponding to nt 1326–1584 within the open reading frame of GenBank entry AB040896 (see Fig. 1) was amplified by PCR using upstream 5'-CGGATCCACAGAAGCATTG-3' and downstream 5'-AGATGGGGTCTAACTGGTCAGC-3' as the primers. This fragment was subcloned into pBluescript II KS(–) vector (Stratagene, La Jolla, CA). Sense and antisense riboprobes were generated by incubation of EcoRI- or BamHI-linearized templates with digoxigenin-labeled uridine 5-triphosphate in the presence of T7 (sense) or T3 (antisense) RNA polymerases according to the manufacturer’s recommendations (Roche Biochemicals, Mannheim, Germany). RNA in situ hybridization was carried out on 5-µm tissue sections according to an established protocol (3) except for the omission of dithiothreitol in the prehybridization buffer and RNase A in the posthybridization washes. The hybridization temperature was 47 C. The sections were counterstained with nuclear fast red for 10 min.
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Polyclonal antibodies were custom made by Bethyl Laboratories (Montogomery, TX). Briefly, the following peptide sequences were synthesized: midpiece peptide, C I D T D D L P R K R L P Q L Y K; and C terminus peptide, C I D I N S R G E K Q R M H L R D. These sequences were chosen using company proprietary software designed to identify antigenic sites within the protein and with low possibility of cross-reactivity to known protein sequences. The peptides were synthesized to contain an N-terminal cysteine, using a Rainen multiple peptide synthesizer, analyzed by mass spectroscopy, and HPLC purified to more than 90% purity. Purified peptide was linked via the sulfhydryl of the N-terminal cysteine to maleimide-activated keyhole limpet hemocyanin (Pierce, Rockford, IL); 100 µg of peptide-conjugated keyhole limpet hemocyanin was injected per animal. This was mixed with complete Freund’s adjuvant (Difco, Sparks, MD) for the initial injection and incomplete Freund’s adjuvant (Difco) for subsequent biweekly injections. Sera from the rabbits were then affinity purified and the potency determined by ELISA.
With both antisera the main band detected by Western blot analysis was 115 kDa. To determine the specificity of the ALLP antibodies, the peptide used to generate the antisera was preincubated with the antiserum (1:4 ratio of antiserum to peptide) and subsequently used in Western blot analysis. The 115-kDa band that was noted in protein lysates of endometrium was virtually undetectable when the antiserum was neutralized with peptide. Furthermore, a dose-related increase in the density of the 115-kDa band was detected by loading greater quantities of protein in the Western blot analysis, again indicating that the 115-kDa band detected by our antiserum in Western blot analysis corresponds to ALLP (data not shown).
Immunohistochemistry
Paraffin sections (4 µm) were mounted, deparaffinized in xylene, and hydrated gradually through decreasing concentrations of ethanol followed by a wash in tap water. The slides were then placed in citrate buffer (pH 6.0) and microwaved to the point of boiling and then left in hot buffer for an additional 30 min for antigen retrieval. Endogenous peroxidase was quenched with methanolic hydrogen peroxide (3%) for 10 min, followed by running tap water rinse to clear. The slides were rinsed three times in PBS (pH 7.4) and incubated with normal goat serum (Biogenex, San Ramon, CA). Subsequently they were incubated with primary antibody (titers ranging from 1:50 to 1:200) in PBS with 0.1% BSA at 25 C for 1 h, followed by three rinses with PBS. The slides were then incubated with CytoScan biotinylated mouse/rabbit link antibodies (Cell Marque, Hot Springs, AR) for 20 min, followed by three rinses with PBS. Chromagen was developed using 3,3'-diaminobenzidine tetrachloride substrate (Sigma, St. Louis, MO) for 5–8 min and after wash in tap water counterstained in Harris hematoxylin for 5 min. Negative control sections were incubated with preimmune serum from the same rabbit that was immunized in place of the primary antibody. In addition, immunoneutralization was carried out by preincubating the midpiece peptide with the midpiece antibody at a ratio of 4:1 (peptide to antibody) at room temperature for 2 h. This preparation was then used instead of the primary antibody and served as an additional control. Photography was performed using an E400 microscope (Nikon, Tokyo, Japan) with H-III photomicrographic equipment (Nikon).
Semiquantitative multiplex RT-PCR
Total RNA was extracted from tissues or cells using STAT-60 reagent (Tel-Test Inc., Friendswood, TX), according to manufacturer’s instructions. Four micrograms of total RNA was reverse transcribed into cDNA. PCR was carried out with the same primers used to amplify the in situ hybridization probe template. Two microliters of the generated cDNA reaction and 3:7 ratio of 18S primers to competimers (Ambion, Austin, TX) were used in the amplification reaction that was carried out with an initial step at 94 C for 5 min, followed by 30 cycles of 94 C for 45 sec, 58 C for 45 sec, 72 C for 1 min, and a final step of one cycle of heating at 72 C for 7 min. The PCR products were electrophoresed in 1.5% agarose gel, stained with ethidium bromide, and photographed. The size of the ALLP PCR product was 259 bp. The relative OD of the bands on the gel was analyzed with DigiDoc 1000 (Alpha Innotech Corp., San Leandro, CA).
Sequence analysis
Initial standard BLAST (4) searches and Prosite motif analysis of a shorter transcript of ALLP did not identify homologous proteins at the level of amino acid sequence, and, therefore, the sequence was analyzed with the fold recognition program 3DPSSM (http://www.sbg.bio.ic.ac.uk/3dpssm). Subsequent searches of the full-length 900-amino acid protein against the nonredundant protein sequences (www.ncbi.nlm.nih.gov/blast) using BLAST was used to identify homologous proteins and related proteins whose three-dimensional structures have been determined (Table 2).
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Cells were harvested in ice-cold radioimmunoprecipitation assay buffer (1x PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) containing 100 ng/ml phenylmethylsulfonyl fluoride and protease inhibitors (Roche Biochemicals). The cell lysates were microcentrifuged at 14,000 rpm for 30 min at 4 C. The protein concentration was measured with a BCA protein assay kit (Pierce). Equal amount of protein were separated by SDS-PAGE and then transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA) over 2 h on ice, and the blots were blocked for 2 h at room temperature in buffer containing 1x Tris-buffered saline, 5% milk, and 0.05% Tween 20 and incubated overnight at 4 C with rabbit anti-ALLP polyclonal antibody diluted at 1:50,000. The blots were washed three times with 1x Tris-buffered saline and incubated with second antibody (horseradish peroxidase-conjugated goat antirabbit IgG) for 1 h. The signal for ALLP was detected by a chemiluminescence detection system (Pierce). Bands corresponding to ALLP protein were determined using DigiDoc 1000 (Alpha Innotech, San Leandro, CA). To ensure equal loading of protein the same membranes were re-probed for glyceryl aldehyde-3 phosphate dehydrogenase (GAPDH) after stripping the ALLP antibody.
Statistical analysis
Comparisons between two groups were done by the Student’s t test. In the case of multiple groups, ANOVA followed by post hoc testing (Tukey) was done. Significance was established at P < 0.05.
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Sequence analysis
A comparison of a partial sequence of the ALLP protein against a library of approximately 8000 three-dimensional structures identified a significant similarity (PSSM confidence value = 1.9 x e–33) with proteins of the luciferase-like fold. The alignment of ALLP to the firefly (Photinus pyralis) luciferase structure given by 3D-PSSM is shown in Fig. 1
. Although the overall sequence similarity is low, the compatibility of the sequence with the luciferase fold is quite strong, and the predicted secondary structure of ALLP matches that of luciferase over large portions of the proteins (Fig. 1).
ALLP seems to be closely related but not identical with other genes found in the GenBank database. The only human transcript that has been formally named is DIP2 (previously designated in a random sequencing study as KIAA0184), a putative homolog of Drosophila disco-interacting protein-2 (DIP2) identified in a yeast two-hybrid screen (5). DIP2 is 77% identical (87% similar) to the ALLP ABO isoform and in the XM-derived N-terminal extension region (see below), 69% identical (83% similar). Another human gene similar but not identical with DIP2 is KIAA0934, with 77% homology (87% identity) to ALLP and 76% homology (87% identity) in the N-terminal extension to DIP2. Mouse homologs of KIAA1463 (ALLP), KIAA0934, and KIAA0184 (DIP2) have also been identified. Additionally, RT-PCR experiments detected this mRNA in reproductive and nonreproductive tissues from sheep, mice, and rhesus monkeys as well (data not shown).
BLAST searches of the GenBank database demonstrated 50% nucleotide identity of ALLP with Caenorhabditis elegans hypothetical protein F28B3.1 (GenBank entry AF003136), 56% identity with unknown Drosophila melanogaster protein encoded by GenBank entry AF246991 as well as similar genes in Anopheles gambiae (EAA05281.1) and Schizosaccharomyces pombe (NP593217.1). Thus, this gene seems to be well conserved across both vertebrate and nonvertebrate evolution.
A BLAST search of the full-length 900-residue-long sequence of ALLP against nonredundant protein sequences revealed a high level of sequence similarity with a large number of eukaryotic and prokaryotic proteins. Nearly 200 entries had significance scores better than 9 x e–4. In addition to the genes identified in the BLAST search of GenBank, above, the protein sequences have significant matches with a large number of fatty acid, aryl and acyl coenzyme A (CoA) synthetases/ligases, and enzymes functioning in nonribosomal peptide synthetases (6) and antibiotic biosynthesis (Table 2).
The BLAST search pinpointed two regions of the ALLP 900-residue protein that correspond to putative AMP-binding domains. Luciferase (7) and other proteins sharing the luciferase fold are members of AMP-binding proteins, which include enzymes such as gramicidin synthetase (8), acetyl CoA synthetase (9), and DhbE (a nonribosomal peptide synthetase) of Bacillus subtilis (10), proteins whose three-dimensional structures have been determined. Based on the sequence and structural similarity with the luciferase family of AMP-binding enzymes, we refer to this 900-residue protein as ALLP.
Tissue distribution
Semiquantitative multiplex RT-PCR and Western blot analysis was used to determine the tissue distribution of the ALLP mRNA. The results are shown in Fig. 2. The figure (top portion) reveals a representative gel with the bar plot summarizing the mRNA data; mRNA was detected in all reproductive tissues analyzed with relatively higher levels found in the endometrium and ovary. The protein data are shown in Fig. 2B. The two antibodies directed against the midportion and C-terminal portion of the protein readily detected ALLP by Western blot analysis. Both antibodies identified a protein of approximately 115 kDa, a substantially higher molecular weight than that predicted from the cloned cDNA. As with the ALLP mRNA, the ALLP protein was detected in all tissues examined, with premenopausal tissues and postmenopausal tissues exposed to exogenous hormones showing higher expression levels of ALLP (Fig. 2B). ALLP mRNA was detected in the female reproductive tissues of mice, sheep, and rhesus monkeys, was abundant in the testes of mice, and was readily detectable in nonreproductive tissues such as the kidney, liver, lung, brain, and spleen of mice and sheep (data not shown).
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Distinct cyclic variation in the endometrial expression of ALLP mRNA was noted during the menstrual cycle (Fig. 3A). The highest mRNA levels were noted in the early secretory phase (d 16–21), with a decrease in expression just before menses. Protein data also confirmed these findings in the endometrium showing significantly greater (P < 0.05) ALLP expression in the mid- to late proliferative phase and the early to midsecretory phase (d 17–24), compared with the early proliferative phase and late secretory phase of the cycle (after d 25) (Fig. 3B). Postmenopausal women taking a continuous regimen of conjugated equine estrogen (0.625 mg daily) and medroxyprogesterone acetate (2.5 mg daily) had greater (P < 0.05) expression of ALLP protein, compared with women not taking hormone replacement therapy (Fig. 3C), evidence that ovarian steroids may regulate ALLP expression in the endometrium.
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In situ hybridization was used to localize the mRNA in the female reproductive tract (Fig. 4). ALLP was expressed in the cytoplasm of all tissues examined. In the endometrium (Fig. 4A), both the glandular epithelium and stroma showed positive staining, although there was more intense staining in the glandular epithelium, compared with the stroma. Inflammatory cells did not stain for ALLP. The glandular staining was concentrated in the apical region of the cell in proximity to the lumen. There appeared to be regional variation in glandular staining with glands in the basalis layer showing more intense staining. The microvascular endothelium in the uterus also stained moderately for ALLP, with significant numbers of vessels showing positive staining. There was also some diffuse myometrial staining for ALLP.
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), the luteinized theca externa cells expressed ALLP mRNA. Expression was also detected in luteinized theca interna and granulosa cells, cortical stroma, and endothelial cells of vessels found in the cortical area. The control slides (sense probe) for both the endometrium (Fig. 4C) and ovary (Fig. 4D) failed to show any signal.
Immunohistochemistry was then used to localize ALLP protein in healthy endometrium (Fig. 4E) and endometrial cancer (Fig. 4F). Using the midpiece-directed antiserum, ALLP protein localization closely matched the mRNA localization with staining concentrated within the apical region of glandular epithelium. Both nuclear and cytoplasmic staining was detected. The luminal epithelium also intensely expressed ALLP. Stromal staining for the protein was more intense than for mRNA. Smooth muscle cells within the myometrium showed diffuse ALLP expression, whereas the fibroblasts failed to stain. Sections of endometrial carcinoma (Fig. 4F) (endometrioid type) showed cytoplasmic staining for epithelial cells in malignant glands. The expression pattern of some glands was not uniform because one population of cells showed strong staining, whereas others only had moderate staining. The intervening fibrous stroma did not stain positively. Substitution of the preimmune serum from the animals before injection of immunogen (Fig. 4G) or immunoneutralization of the antiserum by preincubation with the midpiece peptide (Fig. 4H) eliminated ALLP staining, demonstrating the specificity of the antiserum. We obtained identical results using the antiserum directed to the C-terminal region of ALLP (data not shown).
Tumor expression
The expression of ALLP by Western blot analysis was determined in normal endometrial tissue; cultured primary endometrial cells; and endometrial cell lines including Ishikawa cells, RL952 cells, and human endometrial surface epithelial (HES) cells (Fig. 5). Healthy tissues or primary cultured cells showed lower expression of ALLP as compared with the tumor cell lines. Of interest is that HES cells, which are a human endometrial epithelial cell line derived from spontaneous transformation of isolated endometrial surface epithelial cells from benign proliferative endometrium (11), showed lower expression of ALLP, compared with the adenocarcinoma cell lines (Ishikawa, RL952), but higher than nonmalignant primary cultured cells or benign endometrial tissue (Fig. 5).
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An updated entry for AB040896 subsequently directed us to the NCBI Locus Link page for the human gene KIAA1463 (www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?I=57609). Identity of AB040896 with other expressed sequence tags having substantial additional 5' mRNA sequences was revealed (AK097369, XM 051160), which established an overall mRNA of 6626 nt. This is schematically shown in Fig. 6A. These expressed sequence tags indicate identity with a predicted gene on human chromosome 12 including 22 exons and spanning 52,407 bp. To confirm the expression of mRNAs containing these 5' exons, we conducted RT-PCR with primers based on the 5' ends (start codon) of ALLP (AB040896 isoform), AK 097369 and XM 051160. Figure 6B demonstrates that amplification product was obtained with all these 5' primers. The 5'-RACE experiment described above confirms the presence of the AB0 ALLP mRNA variant, and the RT-PCR in Fig. 6B confirms the expression of the XM ALLP mRNA variant. Because the AK097369 primer is internal to the XM primer, we cannot be certain whether the AK PCR product reflects XM isoform expression. The relative intensity of the AK PCR product appears to be consistently higher than the XM PCR product, which might suggest abundance of the mRNA. The molecular weights of predicted proteins are: ALLP (AB0), 53580 kDa; AK 097369, 84101 kDa; and XM 051160, 98045 kDa. The molecular weight of the XM isoform is closest to the observed band on Western blot, allowing for potential posttranslational modification.
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), strongly suggesting that ALLP represents a novel class of enzymes in mammals whose biological functions remains to be identified. Preliminary structural analysis provides very strong evidence that ALLP is an enzyme in which a covalent intermediate with AMP is a feature of the reaction. Several findings suggest that ALLP may have an important physiological function in mammals: 1) ubiquitous expression of ALLP in reproductive and nonreproductive tissues, in human circulation (data not shown), and across species suggests it may play an important role in maintenance of cellular homeostasis; 2) the expression profile of the ALLP transcript during the menstrual cycle suggests that it is regulated by ovarian steroids. The greater expression of the mRNA and protein just before the expected time when implantation occurs suggests a role for this protein in establishment of a hospitable uterine environment; 3) the higher endometrial expression of ALLP in women receiving HRT provides additional evidence for regulation by steroids in this site; 4) the increased expression of ALLP protein in endometrial cancer cell lines, compared with primary cells and normal endometrium, suggest a potential role in regulation of cell division; and 5) the differential localization of ALLP in the uterus and ovary suggest physiological importance in specific compartments.
Analysis of the 2107 bp in the gene representing the potential promoter and upstream enhancer region using the MatInspector software (12) reveals potential binding sites for a number of transcription factors including a TATA-like box. These also include the estrogen receptor at position 558–576 from the start codon site, which supports our data showing cyclical changes in endometrial expression of ALLP mRNA and protein. A possible progesterone receptor binding site was also identified. Other binding sites for potential transcription factors for the ALLP gene are cAMP response element-binding protein, nuclear factor-
B, aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator heterodimers, hypoxia-inducible factor-1
, and the glucocorticoid receptor.
PCR and RACE analysis revealed at least two species of mRNA transcripts, and a third transcript has been deposited in GenBank. This is likely due to alternative splicing because examination of the human genome database did not provide any evidence for the existence of duplicated genes, which are differentially spliced. The AB0 and XM mRNA isoforms of ALLP would encode proteins of substantially different sizes. Despite the predicted molecular weight of ALLP(53.6 kDa), a molecular weight of approximately 115 kDa was detected by Western blot analysis. This suggests that the greater than 6-kb ALLP mRNA isoform is the only translated mRNA. The ALLP mRNAs were unfortunately not detectable by Northern blot analysis; thus, we cannot determine the relative abundance of the mRNAs. The slightly higher molecular weight of the observed band from the predicted protein could be related to posttranslational processing. Alternatively the 115-kDa protein represents a homodimer with glycosylation, and additional studies are needed to clarify this issue. The peptides that were designed for generation of antibodies were not found to have sequence homology to any other known proteins; thus, it is unlikely that the antibody detects proteins other than ALLP.
Sequence comparison of ALLP to published proteins showed clear homology with AMP binding proteins, and the sequence is compatible with the fold of firefly luciferase. Other enzymes related to firefly luciferase include enzymes involved in the biosynthesis of iron-binding siderophores, the antibiotics gramicidin S, tyrocidine and penicillin, and fatty acid/acyl CoA ligase (EC 6.2.1.3) (8, 13). Whereas there are many other adenine and AMP binding protein families that share a common framework made of a tripeptide plus three additional residues that recognize the Watson-Crick edge of adenine (14), the enzymes listed above recognize AMP using a different mechanism whereby the Hoogsteen edge is key to recognition (14).
The question that arises is what the physiological function of a luciferase-like enzyme may be in mammalian cells. The physiological function of protein homologous to ALLP in lower species reveals one group of these proteins, the disco-interacting proteins 1 and 2, both of which are members of the AMP-binding proteins (5, 15). These proteins have been isolated in a yeast interaction trap screen using the protein disconnected (disco) as a bait (5, 15) in Drosophila, human, and mouse. These proteins are developmentally regulated and play important roles in differentiation of the brain. Mutation in the disco gene results in disruption in the connectivity of the larval visual system and differentiation of the circadian pacemaker (16). Overexpression of DIP1 causes the formation of supernumerary structures in the head capsule (17). We found extensive expression of ALLP in embryonic mouse neural tissues (our unpublished data), suggesting a potential role in nervous system differentiation. On the other hand, the significant similarity of ALLP to acyl CoA synthetase and its ubiquitous expression suggests a metabolic function for this protein.
Variations in the expression of ALLP during the menstrual cycle with up-regulation during the early secretory phase suggest that endogenous ovarian steroids may be involved in the regulation of expression of this protein. This is further substantiated by our finding that HRT increases the expression of ALLP in the endometrium of postmenopausal women. In agreement with this concept, analysis of the putative promoter region of ALLP by the MatInspector program (12) showed potential binding site for the estrogen and progesterone receptors. Significant changes in the metabolic activity of the endometrium occur in preparation for implantation, and ALLP may play a role in this function.
In the case of tumor cells, we found progressively increased expression of ALLP in spontaneously transformed endometrial cell line (HES) to adenocarcinoma cells (Ishikawa and RL952 cells). These results suggest involvement of this gene in cell division. Analysis of the putative promoter of ALLP shows binding site for the tumor suppressor P53. Ongoing studies designed to block transcription of ALLP and examine biological end points should provide insight into the physiological function of ALLP.
In summary, we have identified a protein in the female reproductive tract that is a member of the AMP binding proteins and shares homology with firefly luciferase. There are multiple ALLP mRNA transcripts, but based on the size of the protein detected in Western blot analysis, the predominantly transcribed mRNA corresponds to the GenBank entry XM 051160. ALLP is ubiquitously expressed in reproductive and nonreproductive tissues, in human circulation and across species lines. In the endometrium ALLP appears to be regulated by ovarian steroids as distinct changes in mRNA and protein expression is found. The overexpression of ALLP in endometrial cell lines further point to an important role in regulation of cellular metabolic activity and division.
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Acknowledgments |
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Footnotes |
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Abbreviations: ALLP, AMP-binding/luciferase-like protein; CoA, coenzyme A; DIP2, disco-interacting protein-2; GAPDH, glyceryl aldehyde-3 phosphate dehydrogenase; HES, human endometrial surface epithelial cell line; HRT, hormone replacement therapy; iNOS, inducible nitric oxide synthase; RACE, rapid amplification of cDNA ends.
Received January 6, 2004.
Accepted August 20, 2004.
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