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Eppley Institute for Research in Cancer and Allied Diseases (A.K.M.-L., E.B., R.W.R.); Departments of Biochemistry and Molecular Biology (A.K.M.-L., R.W.R.), Obstetrics and Gynecology (J.R.), and Pharmacology (E.B., R.W.R.), University of Nebraska Medical Center, Omaha, Nebraska 68198; and Women’s Services Professional Corporation (C.J.L.), Omaha, Nebraska 68132
Address all correspondence and requests for reprints to: Dr. Raymond W. Ruddon, Corporate Office of Science and Technology, Johnson & Johnson, 410 George Street, New Brunswick, New Jersey 08901. E-mail: rruddon{at}corus.jnj.com
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Abstract |
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hCGß apparently evolved from gene duplication events of the LHß gene. Although the nucleotide sequences of hCGß and LHß are 94% homologous (4), at the amino acid level there is 85% homology between hCGß and LHß over the first 114 of 121 amino acids of LHß (5). There are slight differences in the promoter regions of the genes, which may account for their differential tissue expression, i.e. LHß expression in the pituitary and hCGß expression in the placenta.
Genes 5, 3, and 8 give rise to identical proteins. The gene 7 product differs by one amino acid from the others, a Pro to Met at codon 4 (3). Genes 1 and 2, due to 5'-splice differences, give rise to unique open reading frames that may result in proteins with functions different from those of the other hCGß gene proteins (2). There are also differences among the individual hCGß genes in the promoter regions that may play a role in the differences in expression among these genes. We were interested in determining whether the individual hCGß genes were up- or down-regulated during developmental stages in early pregnancy, perhaps yielding an expression pattern unique to each phase of early embryo growth. To begin to answer this question, we obtained placentas of various gestational ages and screened for hCGß expression of genes 5, 3, 8, and 7.
It is also possible that unusual hCGß expression patterns play a role in the etiology of some clinical conditions, or that they could serve as unique biochemical markers in such cases as spontaneous abortion, blighted ovum, hydatidiform mole, choriocarcinoma, or ectopic hCG-producing cancers. For example, bladder cancers were recently found to produce hCGß, and the level of hCGß gene expression from genes 5, 3, and 8 correlated with the stage of the tumor (6). Transcription levels of these three hCGß genes increased with the stage of disease, whereas only ß gene 7 was expressed in normal urothelial tissue (6).
In the study reported here, the levels of expression of ß genes 5, 3, 8, and 7 were examined in 27 normal placentas of 6–16 weeks gestational age as well as in tissue samples obtained from spontaneous abortion, blighted ova, hydatidiform mole, and cultured JAR choriocarcinoma cells. In most of the normal placentas, gene 5 was the most highly transcribed gene, but there was significant variation in the percentage that gene 5 contributed to the total hCGß gene transcription. There were also highly variable contributions of genes 3 and 8 to the total amount of hCGß messenger RNA (mRNA) produced. Moreover, although the overall patterns of hCGß gene expression varied among the normal placentas, there was no correlation with gestational age. The pattern of expression among the abnormal tissues was not unique and was also seen in 1 or more of the normal placentas. These data suggest that a functional level of hCGß subunit can be obtained by expression of various ß genes and that it is the total level of hCGß produced rather than the expression of individual ß genes that is important for maintenance of early pregnancy.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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RT-PCR
The RNA PCR strategy and subsequent Genescan analysis were
adapted and modified slightly from the methods of Lazar et
al. (6). Contaminating DNA was digested away from the total RNA
with ribonuclease-free deoxyribonuclease I, and the resulting products
were quantified by measuring absorption at 260/280 nm ratios. The first
strand complementary DNA (cDNA) was synthesized from 1 µg total RNA
isolated from tissue, with Superscript II (Life Technologies) at a
final concentration of 2.5 U in the Life Technologies first strand
buffer containing 25 mM Tris-HCl (pH 8.3), 37.5
mM KCl, 4 mM MgCl2, 4
mM dithiothreitol, 0.5 mM of each deoxy-NTP,
and 2.5 µM of random hexamers. Incubation was performed
at 42 C for 50 min, followed by 70 C for 15 min. RNA was then digested
with ribonuclease H at 37 C for 20 min. The PCR reaction mixture
totaled 50 µl and consisted of 5 µl first strand reaction product,
2.5 U Taq polymerase (Life Technologies), and PCR buffer
solution (Life Technologies) containing 20 mM Tris-HCl (pH
8.4), 50 mM KCl, 1 mM MgCl2, 0.2
mM of each deoxy-NTP, and 10 pmol of each of the
hCGß-directed ß1 and ß2 oligonucleotide primers or the
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense and antisense
primers. Oligonucleotide primers were synthesized by the Eppley
Institute Molecular Biology Core Laboratory on an Applied Biosystems
(Foster City, CA) model 394 DNA/RNA synthesizer. hCGß
oligonucleotides were designed (Fig. 1
)
based on data from Lazar et al. (6). Both ß1 and ß2
primers completely matched the flanking sequences of each of the hCGß
genes 5, 3, 8, and 7, resulting in similar amplification efficiencies
for the ß genes, amplifying them proportionally to their transcript
ratios (7).
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. hCGß and GAPDH cDNA fragments
were amplified from the cDNA templates in a Perkin-Elmer (Foster City,
CA) model 2400 thermal cycler using a hot start of 94 C for 4 min,
followed by 35 cycles of 94 C for 30 sec, 60 C for 30 sec, 72 C for 30
sec, and a final extension of 72 C for 7 min.
Genescan analysis
Fifteen microliters of the RT-PCR products with 7.5 µl methyl
green/yellow food coloring-50% glycerol were electrophoresed on a 1%
(wt/vol) agarose gel in Tris-glacial acetic acid-EDTA buffer. The gel
contained 0.2 µg/ml ethidium bromide to visualize the amplified
hCGß and GAPDH amplicons. The hCGß RT-PCR products (17.25 µl)
were digested with 2.5 U DraI and 5 U HhaI (New
England Biolabs, Beverley, MA). Figure 1A illustrates the mRNA
structures of the four hCGß genes under examination and the locations
of the ß primers. DraI and HhaI restriction
sites are labeled, and resulting size fragments are indicated. Digested
DNA fragments were then coelectrophoresed with the Genescan 500
6-carboxy-X-rhodamine (ROX) size standards on a denaturing 6%
acrylamide gel run on a 373 ABI sequencing apparatus by the Eppley
Institute Molecular Biology Core Laboratory. The gel was analyzed by
672 Genescan software, which determines the length of the fragments in
each lane, calculates the intensity of the 6-carboxyfluorescein dye in
the ß2 primer, and expresses the absorbance intensity in relative
fluorescence units. The data are then expressed as an
electrophoretogram displaying the gel DNA bands as peaks that
illustrate the intensity of the dye-labeled primer for each hCGß gene
in terms of peak height and area.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). These
PCR products were then digested with restriction enzymes
DraI and HhaI, which gave different sized
fragments for each gene: ß5, 341 bp; ß3, 360 bp; ß8, 233 bp; and
ß7, 391 bp (Fig. 1A). The smaller restriction products were not seen
because only the ß2 primer, incorporated into each large restriction
product, was fluorescein labeled. The bands on the 6% acrylamide gel
were converted to peaks by the Genescan 672 analysis software, and the
fluorescent peak intensity was calculated for each gene.
Examples of some of the different types of gene expression patterns are
illustrated in Genescan electrophoretograms in Fig. 2, A–D. Figure 2A depicts the 5 >
3 = 8 >> 7 gene pattern detected by Bo et al. (2).
Figure 2B depicts a 5 > 8 >> 3 = 7 gene expression
pattern, Fig. 2C shows a 5 = 3 = 8 >> 7 pattern, and Fig. 2D shows a 5 = 3 > 8 >> 7 pattern. Figure 2D has a unique
peak at approximately 45 bp that is probably a primer dimer or a
nonspecific amplification artifact in this particular sample. To
control for possible genomic DNA contamination of RNA, samples were
also run without Superscript reverse transcriptase, and no
amplification of hCGß was detected by Genescan analysis (Fig. 2E).
The small peaks detected in this panel are the Genescan 500 ROX
internal lane size standards.
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).
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. The mean percent
expression for genes 5, 3, 8, and 7 and the SD for each are
shown in Table 1 for each of the 27
placentas.
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Several other tissues from different clinical conditions, such as
hydatidiform mole, blighted ovum, and spontaneous abortion, were
analyzed to determine their hCGß gene expression (Fig. 5). The hydatidiform mole displayed
hCGß gene expression pattern 5 > 8 >> 3 = 7. Two
blighted ovum specimens were analyzed and found to display a 5 >
8 >> 3 
7 pattern of hCGß gene expression. Although these
samples showed low levels of gene 3, the levels were within the range
of expression observed in the normal placentas. It may be noteworthy
that in one of the blighted ovum samples, hCGß gene 7 was responsible
for 6% of total hCGß expression. This was the highest gene 7
expression detected in any of the samples we examined. The
spontaneously aborted placenta also had a normal pattern of gene
expression: 5 > 3 = 8 >> 7. The JAR choriocarcinoma cell
line was also analyzed to determine which hCGß genes were
transcribed, and a gene expression pattern of 3 > 5 > 8
>> 7 was detected (Fig. 5). Although JAR cells expressed a higher
percentage of total expression from gene 3 and a lower percentage from
gene 5 than was seen in most of the normal placentas, the expression
pattern, 5 = 3 > 8 >> 7, was not unusual. The mean
percent expression for genes 5, 3, 8, and 7 and the SD for
each of the clinical specimens are shown in Table 1.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Our results indicate that normal placentas express genes 5, 3, and 8 in amounts detectable by RT-PCR. However, the expression levels of each gene seem to vary among individuals. In all placentas, hCGß gene 5 was highly expressed, ranging from 35–82% of the total hCGß, but in many cases genes 3 and 8 were also responsible for high levels of expression. Transcription of gene 3 was quite variable, ranging from 1–42% of the total ß gene expression. In two placental samples, gene 3 accounted for only 1–2% of the hCGß expression, whereas in 17 samples it accounted for 25% or less of the ß gene transcript. Gene 8 transcription varied from 12–32% of the total. Gene 7 was also transcribed in detectable levels, except in one 8-week-old tissue specimen that displayed no gene 7 transcript. These data indicate that there is heterogeneity in the levels of expression of each gene in individual placentas. Thus, it appears that the important factor during the first trimester of pregnancy is the total amount of hCGß gene transcription and not necessarily from which gene the mRNA is derived. As the amount of hCGß protein during pregnancy is regulated at the transcriptional level (9), it is likely that the amount of ß gene transcripts correlates with the amount of hCGß protein in the placentas examined in this study.
We were also interested in determining whether the six hCGß genes were being up- or down-regulated with increasing gestational age. No clear pattern of ß gene transcription could be correlated with placental age. The later age placentas appear to have the same heterogeneous gene expression as the earlier age placentas. There does, however, appear to be a slight shift in regulation of hCGß gene 3 as gestational age increases. Some samples of 11 weeks or older showed very low levels of gene 3 expression, and this was not detected in any of the 6- to 10-week-old placentas. More placentas would need to be analyzed to confirm this as a normal event during pregnancy.
To investigate the question of whether atypical patterns of hCGß gene expression occur in some disease states, specimens from the JAR choriocarcinoma cell line, hydatidiform mole, blighted ovum, and spontaneous abortion tissues were examined. These data were then compared with the normal pregnancy data. The hydatidiform mole is a gestational trophoblastic tumor that originates from placental tissue (10). Blighted ovum pregnancies are those that do not develop a fetal pole for unknown reasons (11). If these specimens were to display unusual patterns of hCGß gene expression, then analysis of these patterns could have diagnostic significance. For example, if hCGß gene expression and tumor stage could be correlated for hCG-producing malignancies, then analysis of surgical or biopsy samples could be used to predict prognosis or response to therapy. Similarly, ß gene expression patterns in abortuses might reveal some defect in gene expression in individuals experiencing frequent spontaneous abortions. However, none of the abnormal specimens examined had a pattern of ß gene transcription outside of the variation seen in normal placentas. Further studies on a larger number of specimens need to be conducted to determine whether unusual hCGß gene expression correlates with a particular clinical condition.
There are several possible explanations for the differential expression of the hCGß genes in individuals. First, the nucleotide sequence differences among the promoters for each of the four hCGß genes appear be in areas that make them stronger or weaker promoters (12). Chimeras formed by adding either a portion of the gene 7 promoter to gene 5, or the gene 5 promoter to gene 7 display no activity or full activity, respectively, when transfected into Y1 cells (12). This suggests that it is the nucleotide differences in the promoter region from MstII (-187) and SpeI (+103) that are partially responsible for the disparity in transcription levels (12). A number of putative transcription factor-binding sites have been identified in this region of the hCGß promoters. There are two cAMP regulatory element-binding sites (13, 14), three to four trophoblast-specific element (TSE)-binding sites that would aid in activation of the hCGß genes (15), and an Oct 3/4-binding site that is probably involved in repression of hCGß gene transcription in during early stages of pregnancy (16). There are two putative Sp-1-binding sites with the sequence GGGAGG in this portion of the proximal promoter region of gene 5. Sp-1 is hypothesized to play a role as a tether to anchor basal transcription factors in the initiation complex of TATA-less promoters, such as hCGß (4, 17). The hCGß genes differ in their nucleotide sequences in these regions, and perhaps this is the cause of the differential expression among the genes. For example, hCGß 7 differs by three nucleotides from gene 5 in the first two TSE-binding sites. The only other differences in binding site regions occur in the Sp-1 sites. As the TSE regions are believed to be only weak activators of the hCGß genes, it may be the changes in the Sp-1 sites that are important for transcription. If these binding sites are indeed used by transcription factors in vivo, then it may be possible that these base changes are responsible for the differential expression of these genes. Mutational analysis experiments have been performed in some of these potentially critical areas. For example, by mutating wild-type hCGß gene 5 from GG at -55 and -54 to TT, which is the native sequence for gene 7, transcription is down-regulated 5-fold (17). Other unique polymorphisms may exist in the population in these regions that also play roles in differential expression of the hCGß genes. Polymorphisms may create better or worse recognition binding sites for the transcription factors, resulting in unique levels of expression in individuals. There is also a distal promoter region (-3700 to -1700) upstream from the proximal promoter in the hCGß genes that plays a role in the expression of the ß genes. Deletion mutants in this region have been shown to affect hCGß basal transcription (1, 4). Nucleotide changes in these distal promoter regions may also change the amount of expression among individuals. Another possible reason for the differential hCGß expression among individuals is that differences in methylation can up- or down-regulate transcription of particular hCGß genes, and perhaps different individuals have unique amounts of methylation in the promoter regions of these genes. It will be of interest to examine a random population of people to gain information on the polymorphisms that exist in the promoter regions and to examine them for methylation differences.
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Acknowledgments |
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is
also gratefully acknowledged. |
Footnotes |
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2 Recipient of Skala and McDonald Fellowships and currently the
recipient of an Emley Fellowship.
Received May 28, 1997.
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References |
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and ß subunits reflects the
steady state levels of the corresponding mRNAs. J Biol Chem 258:9250–9253-subunit gene. Mol Endocrinol 3:1070–1076[Abstract]
and ß genes through distinct types of CREs. J Biol Chem 269:31090–31096- and ß-subunit genes of human
chorionic gonadotropin by trophoblast-specific element-binding protein.
Mol Endocrinol 7:1579–1588[Abstract]
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