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[Cancer Research 59, 1096-1101, March 1, 1999]
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[Cancer Research 59, 1096-1101, March 1, 1999]
© 1999 American Association for Cancer Research

Molecular Biology and Genetics

G-Protein {gamma} 7 Is Down-Regulated in Cancers and Associated with P 27kip1-induced Growth Arrest

Kohei Shibata, Shinji Tanaka, Takeshi Shiraishi, Seigo Kitano and Masaki Mori1

Department of Surgery, Medical Institute of Bioregulation, Kyushu University, Beppu 874 [K. S., S. T., T. S., M. M.], and Department of Surgery I, Oita Medical University, Oita 875 [S. K.], Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously identified and cloned human G protein {gamma} 7 (G-{gamma} 7) gene, which is down-regulated in pancreatic cancer. We examined G-{gamma} 7 expression in other gastrointestinal tract cancers. In 24 of 30 patients with gastrointestinal tract cancer, Northern blot assay and immunohistochemical staining revealed significantly lower G-{gamma} 7 expression in tumors than in normal tissues from the same patients. Semiquantitative reverse transcription PCRs also showed lower G-{gamma} 7 expression in tumors than in corresponding normal tissues in 69 of 90 patients. To examine the biological role of G-{gamma} 7 in cancer, the G-{gamma} 7 cDNA was transfected into a human esophageal carcinoma cell line, KYSE150, that lacks G-{gamma} 7 expression. G-{gamma} 7 expression suppressed cell growth and tritiated-thymidine uptake when cells were confluent. G-{gamma} 7 expression also suppressed tumorigenicity in BALB/c nude mice until 3 weeks after transplantation. G-{gamma} 7 expression increased the G0/G1 population and decreased the S phase population when cells were at high density. We confirmed that this change was associated with p27Kip1 expression. These findings suggest that human G-{gamma} 7 is associated with p27kip1-induced growth arrest and may be a therapeutic target in cancers.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Heterotrimeric G protein, which is composed of {alpha}, ß, and {gamma} subunits, transduces signals across the plasma membrane from a receptor to an effector (1, 2, 3, 4) . Signal transducing elements of the G protein are not only the {alpha} subunit that binds and hydrolyzes guanosine 5'-triphosphate, but also the ß{gamma} subunit, which plays a major role in signal transmission (5, 6, 7) . The G protein ß{gamma} subunits clearly control signals involved in cell growth, but there is no evidence for mutations or alterations of the molecules in human tumors (8) . Whereas signal alteration mediated by small G proteins such as ras (9) , rho (10 , 11) , and rac (10) have been reported in various cancers, the changes in mediated signals of heterotrimeric G proteins are unknown in detail. We have previously reported that human G-{gamma} 72 is down-regulated in pancreatic cancers and cell lines (12) . G protein {gamma} subunits determine the functional specificity and stabilize the heterotrimeric G protein to the cellular membrane (2 , 13, 14, 15, 16, 17, 18) . Because G-{gamma} 7 was expressed in a variety of tissues and may regulate widely distributed signal transduction pathways (19, 20, 21) , the G-{gamma} 7-coupled G proteins might contribute to carcinogenesis in many kinds of cancers. To determine the role of G-{gamma} 7 in carcinogenesis, we herein report status of G-{gamma} 7 expression in other gastrointestinal tract cancers and biological effects of G-{gamma} 7 on malignant phenotypes in a carcinoma cell line. Our study demonstrated that G-{gamma} 7 expression was associated with cell-cell contact-induced growth arrest; thus, we further studied expression of cyclin-dependent kinase inhibitors that link contact inhibition to cell cycle arrest (22 , 23) .


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
RNA Extraction.
Human gastrointestinal tract cancer and corresponding normal tissues were obtained from 90 surgical patients (30 esophageal, 30 gastric, and 30 colorectal). These tissues were immediately frozen in liquid nitrogen after resection, and kept at -90°C until use. Total RNA was prepared by a modification of the guanidinium thiocynate method and dissolved to 1.0 µg/µl with diethyl pyrocarbonate-treated water.

RT-PCR.
cDNA was synthesized from 2 µg of total RNA in a 30-µl reaction mixture containing 5 x reverse transcriptase reaction buffer (Life Technologies, Inc., Gaithersburg, MD), 200 µM dNTP, 100 µM solution of randam hexadeoxynucleotide primers, 50 units of RNasin (Promega, Madison, WI), 10 mM dithiothreithol, and 100 units of Moloney leukemia virus reverse transcriptase (Life Technologies, Inc.). The mixture was incubated at 37°C for 60 min, heated to 95°C for 10 min, and then chilled on ice. PCR was carried out in a 20-µl volume containing 10–20 ng of cDNA, chelating buffer (Perkin-Elmer/Cetus, Norwalk, CT), 200 µM dNTP, {alpha}32P dCTP (Amersham Corp.) at 3000 Ci/mmol, 1.5 units of Taq DNA polymerase (Perkin-Elmer/Cetus), and 0.5 µM of the following G-{gamma} 7-specific primer pairs: up (5'-CCCCAGAGTGATGGCAGACAA-3') and down (5'-TTTGGGGACTTGAGATGTTTTG-3'). The PCR was processed at 94°C for 1 min, 54°C for 1 min, and 72°C for 1 min. To ensure that the RNA was sufficient purity for RT-PCR, a PCR assay with primers specific for the gene GAPDH cDNA was carried out in each case with the same PCR process ((12 , 24, 25, 26, 27) ). GAPDH-specific primer pairs were as follows: up (5'-GTCAACGGATTTGGTCTGTATT-3') and down (5'-AGTCTTCTGGGTGGCAGTGAT-3'). The PCR products were electrophoresed on a 5% nondenaturing polyacrylamide gel. The gel was dried and exposed to an imaging plate, and then the radioactivity was determined using Bioimage Analyzer (Bas1000; Fuji, Kanagawa, Japan).

Northern Blot Assay.
Total RNA (40 µg) was electrophoresed using 1% agarose gel. The RNA was blotted onto a nylon membrane, Hybond N+ (Amersham, Tokyo, Japan) and fixed to the membrane using Stratalinker UV cross-linker (Stratagene, La Jolla, CA). cDNA probe was purified from agarose gel using the QIAEX II gel extraction kit (Qiagen, Chatsworth, CA) and labeled with {alpha}32P dCTP (Amersham) by random primed labeling. Hybridization was performed overnight at 42°C, and the blots were washed with 1 x saline-sodium phosphate-EDTA/0.25% SDS for at least 30 min. The blots were analyzed using BioImage Analyzer (Bas1000; Fuji).

Immunohistochemical Staining.
All samples for immunohistochemical staining were fixed in buffered formalin, embedded in paraffin, and cut in 5-µm thickness. The G-{gamma} 7 protein was detected using antibovine G-{gamma} 7 provided by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), followed by the streptavidin-biotin-peroxidase method (LSAB Kit; DAKO, Kyoto, Japan; Refs. (12 and 28 )).

Cell Line and Stable Transfection.
KYSE150, a human esophageal carcinoma cell line, was provided by Dr. Shimada (First Department of Surgery, Faculty of Medicine, Kyoto University, Kyoto, Japan). KYSE150 was cultured in DMEM supplemented with 10% fetal bovine serum for the following studies. G-{gamma} 7 expression vector, pcDNA3-G-{gamma} 7, was constructed by ligating the G-{gamma} 7 open reading frame into a mammalian expression vector pcDNA3 (Invitrogen, Carlsbad CA). pcDNA3-G-{gamma} 7 was transfected by liposome transfection using Lipofectamine reagent (Life Technologies, Inc., Tokyo, Japan). The exponentially growing KYSE150 cells in a 30-mm dish were incubated for 5 h with Opti-MEM mixture containing pcDNA3-G-{gamma} 7 and Lipofectamine reagent. After 24 h, the cells were subcultured into a 100-mm dish with G418-containing medium. After 2 weeks, several colonies were transferred separately to an individual well of 24-well plates and established as transfectants. G-{gamma} 7 expression in the transfectants was confirmed by RT-PCR and immunoblotting. Primer pairs for the RT-PCR are as follows: up (5'-CAGCCACTAACAACATAGCC-3') and down (5'-TTAAAGGGGTTCGAGGC-3').

Cell Growth Curve.
Cells were seeded at 2 x 105 cells in a 30-mm dish and counted in duplicate at 24-h intervals after plating. The viability of cells was estimated by the dye exclusion method after staining with 0.4% trypan blue solution.

Tritiated-Thymidine Uptake.
Cells were seeded and cultured in 96-well plates at a density of 1 x 104 cells/well for proliferating condition and at a density of 3 x 104 cells/well for 100%-confluence. Tritiated-thymidine (1 µCi; NEN, Boston, MA) was added to each well. After 12 h, at the end of the incubation period, the cells were frozen, thawed, and filtered through glass fiber filters. The ß emission of bound molecules was measured in a scintillation counter.

Tumorigenicity Test in Nude Mice.
Cells were harvested at 107 cells in 0.2 ml PBS and injected into s.c. tissues on the chest wall of Balb/cAnNCrj, nu/nu (4 weeks of age, female) mice. Tumors were measured every week and excised at 3 weeks after transplantation.

Cell Cycle Analysis.
Cells were seeded at 106 cells in a 10-mm dish. After 48, 72, 96, and 120 h, the cells were stained with propidium iodide mixture supplemented with 0.1% sodium citrate and 0.2% NP40. The samples were examined by flow cytometry on a FACScan (Becton Dickinson Immunocytometry System, Mountain View, CA).

Immunoblot Analysis.
Cells were lysed in the sample buffer [50 mM Tris-HCl (pH6.8), 2% SDS, 6% ß-mercaptoethanol, 10% glycerol, and four to six drips of 1% BPB] for the G-{gamma} 7 immunoblotting, or the cold triton-lysis buffer [50 mM Tris-HCl (pH 7.5), containing 1% Triton, 2 mM EGTA, 10 mM EDTA, 100 mM NaF, 1 mM Na4P2O7, 2 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 25 µg/ml aprotinin, 3.5 µg/ml pepstatin A, and 25 µg/ml leupeptin] for p21cip1 and p27kip1 immunoblotting. 100 µg of protein samples were loaded onto 12% SDS-PAGE gels and transferred onto Trans-Blot Transfer Medium (Bio-Rad, Richmond, CA) nitrocellulose membranes. The membranes were probed with NH2 terminus of anti G-{gamma} 7 (Ac-SATNNIAQARKC), provided by Dr. Asano (Institute for Developmental Research, Aichi Human Service Center, Nagoya, Japan), anti p21cip1 (Transduction Laboratories, Lexington, KY), and anti p27Kip1 (Transduction Laboratories). Refined G protein ß{gamma} 7 protein, as a positive control for immunoblotting, was provided by Dr. Asano. Immunodetection was developed using the enhanced chemiluminescence system (Amersham, Buckinghamshire, England).

Statistical Analysis.
The statistical analysis was performed using the Student’s t test.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Semiquantitative RT-PCR.
To confirm the semiquantitative RT-PCR analysis for G-{gamma} 7 expression, levels of radioactivity were measured for cDNAs synthesized with various numbers of PCR cycles. The levels of radioactivity increased linearly to 28 cycles (Fig. 1)Citation . For the internal control GAPDH RT-PCR, the values of radioactivity increased linearly to 22 cycles using the same cDNA sample.



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  		Fig. 1. Correlation between the radioactivity of PCR products and the number of PCR cycles. Semiquantitative PCR conditions for G-{gamma} 7 (A and B) and GAPDH (C and D) are 28 cycles and 22 cycles, respectively.

 
Northern Blot Assay, RT-PCR, and Immunohistochemical Staining.
To examine G-{gamma} 7 expression in gastrointestinal tract cancers, Northern blot assay, RT-PCR, and immunohistochemical staining were performed on sample from 30 gastrointestinal tract cancers (10 esophageal, 10 gastric, and 10 colorectal). G-{gamma} 7 mRNA was displayed as a 1.5-kb band (Fig. 2)Citation . In 24 of 30 samples, Northern blot assay revealed significantly lower G-{gamma} 7 expression in tumors than in normal tissues from the same patients (Fig. 2)Citation . The RT-PCR (Fig. 2)Citation and immunohistochemical staining (Fig. 3)Citation results corresponded with those of the Northern blot assay. In 69 of 90 patients with gastrointestinal tract cancer, RT-PCR also showed lower G-{gamma} 7 expression in tumors than in corresponding normal tissues.



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  		Fig. 2. Northern blot assay for G-{gamma} 7 and GAPDH in 12 cases with esophageal carcinoma (Lanes 1–4), gastric carcinoma (Lanes 5–8), and colorectal carcinoma (Lanes 9–12) are shown in A and B. T, tumor tissue; N, normal tissue. Note the suppressed expression of G-{gamma} 7 in tumor tissues. Semiquantitative RT-PCR for G-{gamma} 7 and GAPDH in corresponding cases are shown in C and D. The RT-PCR results corresponded with those of the Northern blot assay.

 


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  		Fig. 3. Immunohistochemical staining in three representative cases with gastrointestinal tract cancer. In the cases with esophageal cancer (4), gastric cancer (8), and colorectal cancer (12), G-{gamma} 7 stains strongly in the basal part of normal gastrointestinal epitheliums (4N, 8N, and 12N). Cancer tissues (4T, 8T, and 12T) are stained weakly or deleted. The immunohistochemical staining results corresponded with those of the Northern blot assay. HE, H&E staining.

 
Phenotypic Properties of G-{gamma} 7+ KYSE150 Lines in Vitro.
G-{gamma} 7 expression was decreased in gastrointestinal tract cancer tissues. Therefore, we transfected an expression vector containing the G-{gamma} 7 cDNA into several cell lines including Panc1, PK 1 pancreatic carcinoma (12) , and KYSE150 esophageal carcinoma, that lack G-{gamma} 7 expression. Although any stable transfectant could not be established from pancreatic carcinoma, we obtained several stable clones of KYSE150 after G-{gamma} 7 transfection. We used four different clones, parental KYSE150 and three neomycin-resistant transfectants, in our experiments. Transfectant neo+G-{gamma} 7-KYSE150 lacked G-{gamma} 7 expression, and transfectants G-{gamma} 7+KYSE150A and B had G-{gamma} 7 expression, as shown in Fig. 4, A and BCitation . A cell growth curve is shown in Fig. 5ACitation . There was no significant difference in growth rate between G-{gamma} 7- and G-{gamma} 7+ lines. However, at 100% confluence, the cell count was significantly lower in G-{gamma} 7+ lines than in G-{gamma} 7- lines (P <0.001). Incorporation of tritiated-thymidine was lower in G-{gamma} 7+ lines than in G-{gamma} 7- lines at a density of 3 x 104 cells/well (P <0.001), but not lower at a density of 1 x 104 cells/well (Fig. 5B)Citation . These data suggest that G-{gamma} 7 expression suppresses cell growth in vitro when cells are 100% confluent.



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  		Fig. 4. Immunoblotting and RT-PCR for stable transfectants are shown in A and B respectively. Lane P, refined G protein ß{gamma} 7 as a positive control; Lane 1, parental KYSE150; Lane 2, neo+G-{gamma} 7-KYSE150; Lane 3, G-{gamma} 7+KYSE150A; Lane 4, G-{gamma} 7+KYSE150B; Lane M, marker. G-{gamma} 7+KYSE150A and B have G-{gamma} 7 expression, but parent and neo+G-{gamma} 7-KYSE150 lack G-{gamma} 7 expression.

 


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  		Fig. 5. A, cell growth curves (n = 4). Cell count is significantly lower in G-{gamma} 7+ lines than in G-{gamma} 7- lines at 100% confluence (P <0.001). B, tritiated-thymidine uptake, 1.0 x 104 cells/well and 3.0 x 104 cells/well (n = 6). At a density of 3 x 104 cells/well, tritiated-thymidine uptake is also lower in G-{gamma} 7+ lines than in G-{gamma} 7- lines (P <0.001). Plots show the mean ± SE. Lane 1, parental KYSE150 (G-{gamma} 7-); Lane 2, neo+G-{gamma} 7-KYSE150; Lane 3, G-{gamma} 7+KYSE150A; Lane 4, G-{gamma} 7+KYSE150B.

 
Tumorigenicity Test in Nude Mice.
To confirm the biological effects of G-{gamma} 7 in vivo, we examined tumorigenicity in BALB/c nude mice. As shown in Fig. 6ACitation , G-{gamma} 7- lines consistently formed large, progressively growing tumors after s.c. injection into mice; G-{gamma} 7+lines produced smaller, nonprogressive tumors until 3 weeks after injection (Fig. 6B)Citation . Therefore, expression of G-{gamma} 7 suppressed tumorigenicity in nude mice.



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  		Fig. 6. A, tumor volumes (cm3) are shown (n = 8). G-{gamma} 7- lines consistently formed large, progressively growing tumors after s.c. injection into mice; G-{gamma} 7+ lines produced smaller, nonprogressive tumors until 3 weeks after injection. B, representative tumors at 3 weeks after transplantation. Plots show the mean ± SE. 1, parental KYSE150; 2, neo+G-{gamma} 7-KYSE150; 3, G-{gamma} 7+KYSE150A; 4, G-{gamma} 7+KYSE150B.

 
Cell Cycle Analysis and Expression of Cyclin-dependent Kinase Inhibitors.
G-{gamma} 7 expression suppressed cell growth when cells were confluent and suppressed tumorigenicity in nude mice. To study the molecular mechanism of growth inhibition by G-{gamma} 7 expression, we used cell cycle analysis as described in "Materials and Methods." Cell counts were lower in G-{gamma} 7+ lines than in G-{gamma} 7- lines (Fig. 7A)Citation . Cell cycle analysis is shown in Fig. 7, B and CCitation . In G-{gamma} 7+ cell lines, the G0/G1 population gradually increased from 40% to 73%, and the S phase population decreased from 50% to 22%. In contrast, the G0/G1 population of G-{gamma} 7- cell lines remained stable at 50–60%, and the S phase population was stable at 40% (P <0.001). Because expression of G-{gamma} 7 caused cell cycle arrest at G0/G1 phase, we examined the expression of p21cip1 and p27Kip1, cyclin-dependent kinase inhibitors, that are induced by cell-cell contact and arrest the cell cycle at G0/G1 phase (22 , 23) . Surprisingly, expression of the p27Kip1 was dramatically increased in G-{gamma} 7+ cell lines compared with G-{gamma} 7- cell lines (Fig. 7D)Citation . p21cip1was barely detectable at high cell densities, but there was no difference between G-{gamma} 7+ cell lines and G-{gamma} 7- cell lines. G-{gamma} 7-induced G0/G1 arrest at high cell densities seems to be associated with p27Kip1 protein expression.



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  		Fig. 7. A, cell growth curve for cell cycle analysis (n = 4). Cell counts are lower in G-{gamma} 7+lines than in G-{gamma} 7- lines (P <0.01). B and C, cell cycle analysis (n = 4). At 120 h, in G-{gamma} 7+ cell lines, the G0/G1 population gradually increased from 40% to 73%, and the S phase population decreased from 50% to 22%. In contrast, the G0/G1 population of G-{gamma} 7- cell lines remained stable at 50–60%, and the S phase population was stable at 40% (P <0.001). D shows that expression of p27Kip1 in G-{gamma} 7+ cell lines is gradually increased compared with G-{gamma} 7- cell lines. p21Cip1 expression is barely detectable at 120 h, but there is no difference between G-{gamma} 7+ cell lines and G-{gamma} 7- cell lines. Plots show the mean ± SE. Lane 1, parental KYSE150(G-{gamma} 7-); Lane 2, neo+G-{gamma} 7-KYSE150; Lane 3, G-{gamma} 7+KYSE150A; Lane 4, G-{gamma} 7+KYSE150B.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In normal mammalian cells, as in other vertebrates and invertebrates, cell-cell contact inhibits cell proliferation and induces cell differentiation. The details of this mechanism are not known. One model of this mechanism is the mating pheromone-response pathway of the yeast Saccharomyces cerevisiae (29, 30, 31) . Haploid cells of S. cerevisiae through a G protein-mediated pathway respond to a peptide mating pheromone released by yeast of the opposite mating type. Pheromone-induced growth arrest at G1 is associated with induction and phosphorylation of the FAR 1 protein, a cyclin-dependent kinase inhibitor (32) . Genetic studies have demonstrated that the signal transducer in the mating pathway is the Gß{gamma} subunits (33 , 34) , because deletion or loss of function of either the ß (STE4; (29 , 35) ) or {gamma} (STE18; (36, 37, 38, 39) ) subunit blocks the mating pheromone response. In mammalian cells, there is no evidence at present that heterotrimeric G proteins are involved in cell contact-induced growth arrest. Contact inhibition in mammalian cells has been studied in Mv1Lu mink pulmonary epithelial cells, which can be arrested in G1 by growth to high densities. In contact-inhibited Mv1Lu cells, Cdk2-cyclin E activity is reduced, apparently owing to the activation of the p27Kip1. p27Kip1 is a cyclin-dependent kinase inhibitor that has an L-X-X-P-V-N region (X, arbitrary amino acids), which is also found in the Far 1 protein (23) .

Human G-{gamma} 7 expression is generally decreased in gastrointestinal tract cancer tissues. G-{gamma} 7 is expressed in the basal portion of the mucosal epithelium of the gastrointestinal tract. Stable transfection of a carcinoma cell line with a G-{gamma} 7 expression vector revealed that G-{gamma} 7 suppressed cell growth and tritiated-thymidine uptake when cells were 100% confluent. G-{gamma} 7 expression also suppressed tumorigenicity in nude mice. Cell cycle analysis revealed that G-{gamma} 7 arrest of cell proliferation was associated with p27Kip1 protein expression; this phenomenon was not observed in the parental cell line or G-{gamma} 7- transfectant. p21cip1, a cyclin-dependent kinase inhibitor that is associated with contact inhibition (22) , was barely detectable at high cell densities, but there was no difference in expression levels between G-{gamma} 7+ lines and G-{gamma} 7- lines. These findings suggest that the G-{gamma} 7-coupled heterotrimeric G proteins may transduce a growth inhibition signal with cell contact in normal cells, but this does not occur in cancer by inactivation of G-{gamma} 7.

Our study revealed that G-{gamma} 7 expression induced p27kip1 expression in cancer cells with cell-cell contact. p27kip1 expression was associated with the survival of various carcinoma patients (28 , 40, 41, 42) , suggesting that G-{gamma} 7 may be a therapeutic target for cancer. Additionally, G-{gamma} 7 expression was generally decreased in pancreatic, esophageal, gastric, and colorectal cancer tissues; thus, we expect that a G-{gamma} 7-based gene therapy might be effective in patients with these cancers. Recent analysis revealed that G-{gamma} 7 is located on human chromosome 19p13.3.3 Loss of chromosome 19p13.3 was observed frequently in several cancers, including pancreatic cancer (43) , astrocytoma (44) , and ovarian cancer (45) . Furthermore, STK11, Peutz-Jeghers gene, is also located on the telomeric region of chromosome 19p13.3 (46 , 47) . We plan to investigate G-{gamma} 7-mediated signal transductions, genomic alterations, and G-{gamma} 7-based gene therapy.


   ACKNOWLEDGMENTS
 
We are grateful to Drs. T. Asano, T. Kudoh, K. Mafune, and H. Ueo for helpful discussion and also thank K. Sato, T. Shimooka, J. Miyake, and K. Ogata for excellent technical assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Department of Surgery, Medical Institute of Bioregulation, Kyushu University, 4546 Tsurumibaru, Beppu 874, Japan. Phone: 81-977-27-1650; Fax: 81-977-27-1607; E-mail: mmori{at}tsurumi.beppu.kyushu-u.ac.jp Back

2 The abbreviations used are: G-{gamma} 7, G-protein {gamma} 7; RT-PCR, reverse transcription PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back

3 GenBank accession number F23259; sequence information was obtained at: http://www-bio.llnl.gov/bbrp/genome/genome.html (F23259 was submitted by J. E. Lamerdin et al.). Back

Received 9/21/98. Accepted 1/ 4/99.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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