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*Substance via MeSH
Medline Plus Health Information
*Thyroid Cancer
The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 2 554-559
Copyright © 1998 by The Endocrine Society

Original Studies

Absence of Activating Mutations of the Genes Encoding the {alpha}-Subunits of G11 and Gq in Thyroid Neoplasia1

Matthew D. Ringel2, Motoyasu Saji, William F. Schwindinger, Dorry Segev, Martha A. Zeiger and Michael A. Levine

Division of Endocrinology and Metabolism (M.D.R., W.F.S., M.A.L.) and Division of Endocrine and Oncologic Surgery (M.S., D.S., M.A.Z.), The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Address all correspondence and requests for reprints to: Michael A. Levine, M.D., Division of Endocrinology and Metabolism, The Johns Hopkins University School of Medicine, Room 863, Ross Research Building, 720 Rutland Avenue, Baltimore, Maryland 21205. E-mail: mlevine{at}welchlink.welch.jhu.edu


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 Note Added In Proof
 References
 
Activating mutations of the TSH receptor and {alpha}-subunit of Gs (G{alpha}s) that increase adenylyl cyclase activity have been identified in a subset of hyperfunctioning benign thyroid follicular adenomas and, less commonly, in hypofunctioning adenomas and carcinomas. In addition, some thyroid tumors exhibit inappropriate activation of phospholipase C (PLC), a signaling pathway that has been implicated in the growth and dedifferentiation of thyroid cells. We therefore hypothesized that some thyroid tumors might be caused by somatic mutations in the genes encoding the {alpha}-chain of Gq or G11 that result in constitutive activation of the PLC pathway. We amplified regions of the {alpha}q and {alpha}11 genes that encode amino acids, Q209 and R183, and we screened the DNA for mutations by sequence analysis and denaturing gradient gel electrophoresis. No mutations were identified after analysis of DNA from 38 thyroid tumors and 2 poorly differentiated thyroid carcinoma cell lines, including: 13 follicular adenomas, 10 follicular carcinomas, 5 papillary carcinomas, and 10 hyperplastic nodules from multinodular goiters. We conclude that activating mutations of {alpha}q and {alpha}11 are absent or rare in hypofunctioning thyroid neoplasms and that other mechanisms must explain the elevated PLC activity reported in thyroid carcinoma.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 Note Added In Proof
 References
 
TSH, THE PRIMARY regulator of thyroid gland growth and function, exerts its actions by binding and activating a heptahelical receptor [TSH receptor (TSH-R)] that is capable of stimulating members of all four families of the heterotrimeric guanosine 5'-triphosphate (GTP)-binding proteins (G proteins) (1). TSH-R activation leads to stimulation of adenylyl cyclase and ß-isoforms of phospholipase C (PLC-ß) via coupling of the receptor to Gs and members of the Gq/11 family, respectively (1, 2). Stimulation of adenylyl cyclase increases levels of intracellular cAMP, a second messenger that activates signaling pathways that promote cell growth, proliferation, and function in thyroid and other cells but which maintain cell differentiation (3, 4, 5). Somatic mutations of the genes encoding the TSH-R and G{alpha}s that result in constitutive (i.e. hormone independent) activation of the adenylyl cyclase pathway have been identified in benign hyperfunctioning follicular adenomas (6, 7) and, less commonly, in thyroid carcinoma (8, 9, 10, 11, 12, 13, 14, 15, 16, 17). Moreover, germline activating mutations of the TSH-R have been identified as a cause of congenital, nonautoimmune hyperthyroidism (18, 19). The important role of cAMP in the pathogenesis of these forms of thyroid hyperfunction has been confirmed through the generation of transgenic mouse lines, in which constitutive activation of the cAMP cascade in thyroid follicular cells leads to the development of thyroid hyperplasia and autonomous hyperthyroidism (20, 21, 22). However, the role of the cAMP activation in the development of benign and malignant hypofunctioning thyroid tumors is more controversial (23).

At high concentrations of TSH, the TSH-R is able to activate PLC-ß via members of the Gq/11 family (2). When activated, PLC hydrolyzes phosphatidylinositol, yielding diacylglycerol and inositol phosphates. Inositol triphosphate triggers increases in intracellular free calcium, whereas diacylglycerol stimulates protein kinase C enzymes (24). In the thyroid, Gq-mediated activation of PLC-ß elicits cellular proliferation and dedifferentiation, characterized by decreased thyroglobulin expression and reduced iodine uptake, features that often are associated with aggressive forms of thyroid cancer (25). These studies complement recent observations that levels of PLC activity are greater in plasma membranes prepared from neoplastic thyroid tissue than from normal thyroid glands, and that the level of PLC activity correlates with the degree of tumor dedifferentiation (26, 27). New evidence that the PLC signaling cascade may be involved in the pathogenesis of thyroid neoplasia comes from the generation of transgenic mouse models in which PLC is activated in thyroid follicular cells. In one model, mice that express a constitutively active form of G{alpha}q (Q209L) in thyroid follicular cells develop thyroid hyperplasia that is associated with features that are suggestive of early malignancy (28). In a second model, murine lines that express a constitutively active form of the {alpha}1B-adrenergic receptor that costimulates both adenylyl cyclase and PLC in the thyroid develop malignant lesions more frequently and earlier than mice expressing an adenosine receptor coupled only to activation of adenylyl cyclase (29). Taken together, these data suggest that activation of PLC may have oncogenic potential in thyroid cancer.

To better understand the role of PLC activation in the pathogenesis of thyroid cancer, we isolated genomic DNA from thyroid tumors and analyzed the genes encoding G{alpha}q and G{alpha}11 for mutations that replace amino acids R183 or Q209, thereby inhibiting intrinsic GTPase activity and leading to constitutive activation of the protein. The results demonstrated that these sequences were normal in all thyroid tissues examined, indicating that activating mutations of Gq and G11 at residues Q209 and R183 and the surrounding regions occur infrequently, if at all, in thyroid neoplasia and are unlikely to explain the previously reported elevations in PLC activity in thyroid cancer.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 Note Added In Proof
 References
 
Patients

Surgical tissue samples, containing at least 80% thyroid tumor cells, plus adjacent normal thyroid gland were obtained from patients undergoing thyroidectomy and were immediately frozen in liquid nitrogen and stored at -70 C until analysis. Tumors were classified, using standard histological criteria, by a single pathologist (William H. Westra, M.D.), and included 13 nonfunctioning follicular adenomas, 10 follicular carcinomas, 5 papillary carcinomas, and 10 hyperplastic nodules from multinodular goiters. In addition, 2 poorly differentiated thyroid cancer cell lines, ARO and WRO (the generous gifts of Dr. R. Juillard, University of California, Los Angeles, CA), were analyzed.

Amplification of G{alpha}q and G{alpha}11 genes

Genomic DNA was extracted from tumor tissue and from normal surrounding tissue using TRIzol reagent (Life Technologies, Gaithersburg, MD) as per the manufacturer’s protocol. The ARO and WRO cell lines were cultured, as described (30), using RPMI 1640 medium (Life Technologies), supplemented with sodium pyruvate and sodium bicarbonate and 10% FBS. Cells were grown to confluence, and genomic DNA was isolated using TRIzol reagent (Life Technologies).

In the absence of information about the intron-exon structure of the G{alpha}q and G{alpha}11 genes, oligonucleotide primers were designed to amplify fragments surrounding the R183 and Q209 residues, using the known complementary DNA (cDNA) sequences for human G{alpha}q (31) and G{alpha}11 (32). In some instances, one oligonucleotide primer in a pair was synthesized with a 40-base GC-rich extension at the 5' end to optimize analysis of the amplicon, by denaturing gradient gel electrophoresis (DGGE) (33). The sequences of the primers and the corresponding sequences of the genes are shown in Fig. 1Go.



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  		Figure 1. PCR primers used to amplify the R183 and Q209 regions of G{alpha}q and G{alpha}11. Primers with 5' GC clamps are identified by an asterisk. Panel A, MDR 3 and MDR 4 were used to amplify the G{alpha}q R183 region. The 3' end of MDR 3 corresponds to a deletion in the pseudogene, thus allowing for selective amplification of the G{alpha}q gene. Panel B, MDR 1 and MDR 2 were used to amplify the Q209 regions of the G{alpha}q gene and pseudogene. The primer was based on the gene sequence. Bases that differ between the gene and pseudogene are enlarged and are in italics. There were two base pair differences between the gene and pseudogene in this fragment, one in the MDR 1 primer, and one that converted a Bcl-1 site into a Taq-1 site. Panel C, MDR 43 was used with primer MDR 4 to create an R183G mutant as a positive control for DGGE. The single base substitution is underlined (CGA to GGA) that causes the R183G mutation and also results in the loss of a Taq-1 restriction site. Panel D, MDR 23 and MDR 33 were used to amplify and sequence the G{alpha}11 R183 region, and MDR 53 and 54 were used to amplify and sequence the G{alpha}11 Q209 region.

 
Because a pseudogene for G{alpha}q has been identified (31), we designed primers that would allow selective amplification of the R183 region of the G{alpha}q gene (Fig. 1AGo). However, the nucleotide sequences in the Q209 regions of the gene and pseudogene were too highly conserved to permit design of primers that would allow the selective amplification of the G{alpha}q gene. Consequently, we synthesized primers that would anneal to both gene and pseudogene and which flanked a region in which the gene and pseudogene differed by a single nucleotide. Because this single base difference converted a BclI restriction site in the gene into a TaqI restriction site in the pseudogene, we digested genomic DNA samples with TaqI (Life Technologies) for at least 3 h (50 C, 5 U/µg DNA) before PCR, to cleave the pseudogene template and thereby prevent its amplification (Fig. 1BGo).

PCR was performed using Taq DNA polymerase (Perkin Elmer, Foster City, CA) and the primer pairs shown in Fig. 1Go (0.5 µmol/L each), to amplify genomic DNA (400 ng) in a total reaction vol of 50 µL. A programmable thermal cycler (MJ Research, Watertown, MA) was used to perform 40 cycles of PCR, each cycle consisting of denaturation (94 C, 1 min), annealing (1 min), and extension (72 C, 1 min). In the initial cycle, denaturation was increased to 5 min, and in the final cycle, extension time was increased to 5 min. The annealing temperatures were optimized for each target: 60 C for the R183 region and 62 C for the Q209 region of G{alpha}q, and 65 C for the R183 region and 60 C for the Q209 region of G{alpha}11. A sample, in which DNA was replaced with water, was always included as a negative control. After PCR, DNA was visualized by ethidium bromide staining and ultraviolet transillumination. Amplified DNA products were then analyzed by direct nucleotide sequencing or DGGE.

DGGE of G{alpha}q

DGGE of the G{alpha}q PCR products was performed by electrophoresis at 80 V for 14 h at 60 C through 10% polyacrylamide gels containing a linear increasing concentration of denaturants (100% denaturant consists of 8 mol/L urea and 100% formamide), as previously described (31). Optimal resolution was obtained with gradients of 50–80% for the R183 region and 40–70% for the Q209 region. DNA was visualized by ethidium bromide staining and ultraviolet transillumination.

Positive controls for DGGE analysis of G{alpha}q consisted of a DNA fragment containing a R183G mutation and a murine G{alpha}q cDNA containing a Q209L mutation (the generous gift of Dr. Ravi Iyengar, Mt. Sinai Medical School, New York, NY). To generate the R183 fragment, the wild-type sequence was amplified with primer MDR 4 and a mutagenesis primer (MDR 43) that contained a single base mismatch that abolished an existing TaqI restriction site. Creation of the mutant was confirmed by restriction endonuclease digestion and direct sequencing. Mutant and wild-type PCR products were mixed in a 1:1 ratio and denatured and reannealed (Fig. 2Go) to confirm that a single base change in this region could be detected by DGGE (34). A positive control of the Q209 region was obtained by mixing the mutant and wild-type Q209L cDNA templates that differ by one oligonucleotide in a 1:1 ratio before amplification, using primers MDR1 and MDR2. The identity of the bands was confirmed by isolating homoduplex DNA fragments from DGGE gels and performing direct sequence analysis.



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  		Figure 2. DGGE of G{alpha}q Q209 and R183 regions. A, Wild-type (WT) and positive control R183 mutant PCR products were electrophoresed through denaturing gradient gels. Homoduplex bands are depicted, and the sequences were confirmed by direct thermal cycle sequencing. The two WT products were amplified from thyroid tumor genomic DNA. B, PCR products generated from WT and Q209L mutant murine DNA were analyzed by DGGE. The single base pair difference at the Q209 site was detected. The identities of the homoduplex bands were confirmed by direct thermal cycle sequencing.

 
Sequencing of PCR products

To screen the G{alpha}11 gene for activating mutations, direct thermal cycle sequencing of the PCR products from the two regions was performed using 33P-ddNTPs (Amersham, Arlington Heights, IL) and oligonucleotide primers used in the PCR reactions. Thirty cycles of denaturation (94 C, 45 sec), annealing (60 C, 45 sec), and extension (72 C, 1 min) were completed; in the initial cycle, denaturation was increased to 5 min, and in the final cycle extension was increased to 8 min. PCR products were treated with shrimp alkaline phosphatase and exonuclease I before thermal sequencing (US Biochemical Corp, Freehold, NJ) and were visualized on glycerol tolerant gels as per the manufacturer’s recommendations.

To confirm the identity of G{alpha}q homoduplex DNA fragments that were resolved by DGGE, gel slices containing homoduplex bands were cut from the gels, minced, and incubated overnight in 20 µL water at 37 C. One microliter of the solution was used as DNA template for direct thermal cycle sequencing, as above, or was used as template to generate PCR products using the primer pairs in Fig. 1Go, a and b (without GC clamps), that were subcloned into the TA cloning vector (InVitrogen, San Diego, CA). Plasmid DNA was isolated from several clones (35) and was sequenced using M13 forward and reverse primers and Sequenase enzyme (US Biochemical Corp).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 Note Added In Proof
 References
 
Substitution of residues R183 and Q209 of G{alpha}q and G{alpha}11 inhibit GTPase activity and result in constitutive activation of the G protein. We screened the regions surrounding these codons, for mutations, using DNA from neoplastic and adjacent normal tissue and the two thyroid cancer cell lines associated with increased PLC activity (data not shown). No mutations were identified in any of the samples. Figure 2Go depicts the positive controls for the DGGE analysis of G{alpha}q amplicons; Figs. 2Go and 3Go show examples of DGGE analyses in which only wild-type sequences were present in fragments containing the R183 (Fig. 2Go) and Q209 (Fig. 3Go) codons.



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  		Figure 3. Selective amplification of the G{alpha}q gene Q209 region. To avoid coamplification of the Gq pseudogene, genomic DNA was digested with Taq-1 before PCR amplification. Successful digestion of the pseudogene was confirmed by complete digestion of PCR product with BCL-1 and by DGGE, as shown. Lanes in which the genomic DNA was digested with Taq-1 (+) and those not digested with Taq-1 (-) are identified. Note the loss of the lower homoduplex bands and the more slowly migrating heteroduplex bands with Taq-1 digestion. The identities of the homoduplex bands were confirmed by direct sequencing. The preferential amplification of the gene, relative to the pseudogene, likely reflects the single base pair difference between the pseudogene and the MDR 1 primer sequence.

 
Using gene-specific primers, we were able to avoid amplification of the R183 region of the G{alpha}q pseudogene (Fig. 1Go). Because of similarities between the gene and pseudogene in the Q209 region, we digested genomic DNA with TaqI (Life Technologies) before PCR selectively cleaved the pseudogene template and prevented amplification. To confirm that the pseudogene had not been amplified, PCR products (amplified from Taq-1-digested genomic DNA) were digested with BclI (Life Technologies) for 3 h (37 C, 5 U/µg DNA) and analyzed by electrophoresis. All of the DNA was cleaved, confirming that the amplified products contained the BclI restriction site present in the gene but not the pseudogene (data not shown). Moreover, PCR products (generated from genomic DNA that was not digested with TaqI) demonstrated heteroduplexes on DGGE, whereas only a single homoduplex band [containing the wild-type sequence (confirmed by direct sequencing)] was generated after PCR of TaqI digested genomic DNA (Fig. 3Go).

Regions surrounding the R183 and Q209 codons of G{alpha}11 were amplified and sequenced directly, because these amplicons did not focus well in DGGE gels (data not shown). Figure 4Go, a and b, illustrate the nucleotide sequences of the regions directly surrounding these two codons. No mutations were identified in any of the tumors or cell lines. No pseudogene of G{alpha}11 has been described.



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  		Figure 4. Direct thermal cycle sequencing of the G{alpha}11 R183 and Q209 regions. A, Direct sequencing of the R183 region revealed wild-type (CGC) sequences for each tumor, as exemplified by the three sequences depicted; B, direct sequencing of the Q209 region revealed wild-type sequences (CAG) for all tumors evaluated as exemplified by the four papillary tumor sequences shown.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
Note Added In Proof
 References
 
In the present study, we hypothesized that activating mutations in the genes encoding the {alpha}-chains of Gq or G11 may be present in thyroid carcinoma. Several lines of evidence support this hypothesis. First, expression of GTPase-deficient forms of Gq in rodent cell lines leads to increased PLC activity and can induce malignant transformation (36, 37), although its oncogenic potential may be modest in the absence of coactivation of other mitogenic pathways (28, 36). Second, signaling through heptahelical receptors that are coupled to Gq/11 elicit powerful proliferative and transforming effects (6, 7, 18, 19, 29). Recent evidence indicates that activation of these G proteins stimulates mitogenic signals that are mediated not only by PLC but also by additional signaling pathways (38). And third, PLC and PKC activities have been reported to be elevated in thyroid cancers (26), and protracted stimulation in vitro of the DAG/protein kinase C cascade by phorbol esters can promote growth and dedifferentiation of thyroid cells (25, 27). These physiological consequences of protein kinase C activation would likely lead to a phenotype similar to that present in thyroid carcinoma, a result recently demonstrated in transgenic mice that express an activated form of the {alpha}1B-adrenergic receptor that costimulates adenylyl cyclase and PLC (29).

We were able to successfully amplify and analyze the regions surrounding R183 and Q209 codons of the G{alpha}q and G{alpha}11 genes, and we were able to avoid coamplification of the G{alpha}q pseudogene (see above). Results from DGGE and direct nucleotide sequencing indicated that there were no mutations in these regions of the two genes. Thus, we must conclude that activating mutations of R183 and Q209 occur infrequently, if at all, in thyroid neoplasia. Our results thus extend the recent study by Dong et al. (39), in which activating mutations of G{alpha}q or G{alpha}11 were not found in pituitary thyrotroph tumors, another tissue in which heptahelical receptors are coupled via Gq and G11 to mitogenic signaling pathways.

It is improbable that our failure to detect mutations in these regions of the G{alpha}q and G{alpha}11 genes was related to sensitivity. The techniques we used to screen the G{alpha}q and G{alpha}11 genes for mutations are sensitive and specific when a mutant allele is present in a large proportion of the cells in a tissue sample. Histological evaluation of these tumors indicated that more than 80% of the normal thyroid tissue was replaced by tumor, thus, a somatic mutation would be expected to produce an approximately 1:1 ratio of mutant:wild-type alleles. Moreover, our use of genomic DNA, rather than RNA, enabled us to avoid concerns related to relative abundance of wild-type and potentially mutant messenger RNAs, or to possible differences in the efficiency of reverse transcription of these templates.

DGGE, optimized through the addition of a GC clamp to the amplified DNA fragment, has been shown to detect more than 95% of heterozygous gene mutations (33), including point mutations and many small deletions in genes encoding G proteins (11, 34, 40, 41). The human and murine positive controls that we used demonstrated the ability of DGGE to resolve single-base mutations in the G{alpha}q sequence at both of the target codons. Moreover, using DGGE, we could distinguish between the Q209 fragments generated from the G{alpha}q gene and pseudogene, which differ by only a single nucleotide.

Direct sequencing of PCR products was performed to analyze the two G{alpha}11 fragments. The high quality of the sequencing data generated provides strong assurance that mutations were not present in the G{alpha}11 gene fragments. Any ambiguous sequences were confirmed by sequencing in the opposite direction of the first reaction.

The inability to detect activating mutations in the R183 and Q209 residues raises the likelihood that other mechanisms are responsible for the elevated PLC and PKC activity reported in thyroid tumors. Although our results cannot exclude the possibility that activating mutations may be present in other regions of G{alpha}11 or G{alpha}q (e.g. G48), naturally occurring amino acid substitutions in other codons that result in constitutive activation of these G proteins have not been described (42). It is possible that activating mutations of other members of the Gq/11 family are present in thyroid tumors; however, only Gq and G11 are reported to be expressed in thyroid tissue. Recent work has shown that the TSH-R couples to G{alpha}i, G{alpha}12, and G{alpha}13 in addition to G{alpha}s, G{alpha}q, and G{alpha}11 (1, 2). Because Gi family {alpha}-subunits are known to activate PLC in a pertussis toxin-sensitive manner, it is conceivable that activating mutations in this family of {alpha}-subunits are present in thyroid neoplasia. Indeed, a direct role for constitutive activation of G{alpha}i in mutagenesis is supported by the identification of the GTPase-deficient putative gip2 oncogene in several endocrine tumors (8, 42, 43), and overexpression of wild-type G{alpha}i-1 in a single thyroid tumor (44). Additional studies, correlating PLC activity and regulation to levels of G protein expression and presence mutations, are needed to clarify the role of this pathway in human thyroid neoplastic and nonneoplastic hyperplasia, because these two entities may arise by different mechanisms (45). In fact, monoclonal nodules arising from different clones and polyclonal nodules have been described within the same thyroid gland, emphasizing the potentially wide disparity in the molecular events that result in nodule formation (46).

It is possible that the elevated PLC activity present in thyroid tumors represents activation of isoforms of PLC that are not stimulated by G proteins (47). PLC {gamma} activation, via tyrosine kinase receptors or by downstream members of this pathway (such as ras or MAP kinase) may be responsible for the elevated PLC and PKC activity reported in neoplastic thyroid membranes (48).

In conclusion, GTPase inactivating mutations at Q209 and R183, in the genes encoding G{alpha}q and G{alpha}11 that cause constitutive activation of PLC, are either absent or are rare in thyroid neoplasia. Alternative mechanisms that result in abnormally elevated PLC activation in thyroid carcinoma may be involved in the pathogenesis of thyroid cancer, and further studies are warranted.


   Note Added In Proof
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Note Added In Proof
References
 
While this manuscript was under review, we and others (Bai, Y. H., X. H. Bai, and J. J. Murtagh) discovered the existence of a G{alpha}11 pseudogene (accession number AF011499). The primers that we used to amplify regions of the G{alpha}11 gene do not amplify the corresponding regions of the G{alpha}11 pseudogene.


   Acknowledgments
 
We are indebted to William H. Westra, M.D., for his expertise in histology and to Chang Ling Ding, M.D., for his technical assistance.


   Footnotes
 
1 This work was supported, in part, by United States Public Health Service Grants R01-DK-34281 (to M.A.L.), Clinical Associate Physician Award 3M01-RR-00722–22S1 (to W.F.S.), and RR-00052 to the Johns Hopkins Outpatient General Clinical Research Center. Back

2 Recipient of a Pfizer Postdoctoral Fellowship in Endocrinology. Back

Received July 11, 1997.

Revised September 10, 1997.

Accepted October 9, 1997.


   References
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Note Added In Proof
 References
 

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