mRNA Expression of Three Isoforms of Akt in Different Human Cancer Cell Lines




Wei Duan

Introduction


The serine-threonine protein kinase Akt( PKB) involves in many intracellular activities such as cell proliferation, apoptosis, transcription, cell migration and glucose metabolism. It is a key downstream effector of phosphoinositide 3-kinase (PI3K). The PI3K/Akt pathway seems to be one of the most potent survival signaling pathways. The Akt family has three closely related isoforms Akt1, Akt2 and Akt3 encoded by three different genes located on chromosome 14q32, 19q13 and 1q43. They are widely distributed in human tissues, though Akt3 expression seems to be more restricted, being primarily in brain and testis.

The first evidence leading to a role of Akt in tumorigenesis was given by early studies of transforming murine virus ATK8. In human cancers, Akt deregulated activities could result from several mechanisms: inappropriate activation of the upstream partner PI3K, loss of PTEN, Akt gene amplification and Akt protein overexpression. Overexpression of Akt isoforms are reported in different cancers. One mechanism for Akt isoform increased expresison is gene amplification. Akt1 amplification has been found in gastric cancer (Staal, 1987), Akt2 gene amplification has been found in ovarian and pancreatic cancers, with ten percent of pancreatic cancer cells showing Akt2 amplification (Cheng et al., 1996). Overexpression independent of gene amplification has also been found among different cancers. For example, increased Akt2 kinase activity was described in approximately 40% of primary ovarian cancers (Cheng et al., 1996) and Akt3 mRNA has been shown to be upregulated in certain types of melanoma (Stahl et al., 2004). Moreover, gene amplifications of Akt2 and Akt3 tend to relevant with the more aggressive tumor phenotype. Despite these important observations, to date, our understanding of the distinctive roles of Akt isoforms in tumorigenesis is still limited.

To study the relationship of Akt isoforms with cancer, we detected mRNA levels of each Akt isoform using RT-PCR in six different human cancer cell lines: A549, Caco-2, K562, JMN, LAI-5S and LAI-55N. Results reveal that all three isoforms are present in the cell lines studied. mRNA expression of Akt2 and Akt3 share similar pattern in that both show higher levels in A549 and LAI-5S cell lines. No differential expression is found in Akt1.

Material and methods

Material:

Six human cancer cell lines are used. Neuroblastoma cell lines including JMN, LAI-55N and LAI-5S are obtained from the Laboratory of Neurobiology at Fordham University. JMN is the representative of the I-phenotype, LAI-55N is the representative of the N-phenotype and LAI-5S is the representative of S-phenotype.Three other cell lines including A549, Caco-2 and K562 are obtained from Laboratory for Familial Dysautonomia Research.

Method:

RNA extraction
Total RNA from each cell line was extracted and purified from the cell lysates using the RNeasy® Plus Mini Kit (QIAGEN).

RT-PCR
RT‐PCR was performed using QIAGEN® One-Step RT‐PCR Kit following the instructions. GAPDH was used as the loading control.

Gel Electrophoresis
5μl of each RT-PCR product with 2.5μl of loading dye were seperated on 2% agarose gels.

PCR product purification, gel extraction and sequencing
PCR poducts were purified using QIAquick® PCR Purification Kit. Products were cut from the gel and purified using QIAquick Gel Extraction Kit. Purified PCR products were sent out for sequencing.

Results

RT-PCR using the primers(Figure 1.)was conducted in three human cancer cell lines derived from different tissues. GAPDH is served as a loading control. The A549 cell line was developed through the removal and culturing of cancerous lung tissue. Caco-2 cell line is derived from a colon carcinoma. K562 cells were the first established human immortalized myelogenous leukemia line. Results (Figure 2.) show that Akt1, Akt2 and Akt3 all present in those three cell lines. mRNA levels of Akt1 seems to be similar, while both Akt2 and Akt3 exhibit highest levels in A549 cell line. In addition, Akt3 shows least mRNA expression in Caco-2 cell line. Though lower than the mRNA level shown in A549 cell line, no significant difference is exhibited in Akt2 between Caco-2 and K562 cell lines.

mRNA expressions of Akt isoforms were also studied in three neuroblastoma cell lines: JMN, LAI5-S and LAI-55N. GAPDH is served as a loading control. The three neuroblastoma cell lines are representatives of different phenotypes and have different malignancies. JMN is the most malignant one while LAI-5S has little malignancy. Results from RT-PCR show that, again, three Akt isoforms all present in the above neuroblastoma cell lines(Figure 3.). Though no difference is exhibited in Akt1 among the three neuroblastoma cell lines, Akt2 and Akt3 express at higher levels in LAI-5S cell line. In the JMN and LAI-55N cell lines, Akt2 and Akt3 are not differentially expressed.

Discussion

In summary, all three isoforms are expressed in the six cancer cell lines. Previous reports on Akt isoform expression in different tissues indicate that Akt1 and Akt2 are widely distributed, yet Akt3 is expressed mostly in brain and testis. However, our result shows an unrestricted expression pattern of Akt3. This may be due to the limited tissues characteristics and the increasing cancerous features of the cell lines studied. It is possible that tissue specific expression of Akt3 may be lost in cancer cells. No difference of Akt1 mRNA expression was observed in the six cell lines. mRNA expression of Akt2 and Akt3 share similar patterns. Both present at higher levels in A549 cell line and LAI-5S cell line. In fact, relevant research regarding the role of Akt in lung cancer demonstrated that specific depletion of Akt2 in A549 cells significantly reduce cancerous growth and anchorage independent growth and invasion, as compared to Akt1 depletion (Kim et al., 2010). High expression of Akt2 shown in this project is consistent with its suggested role in oncogenesis in A549 cell line. However, surprising results about Akt expression were shown in neuroblastoma cell lines. Among the three neuroblastoma cell lines, LAI-5S, the least malignant one, shows higher mRNA expression of both Akt2 and Akt3. Considering the tumorigenesis potential of Akt isoforms, why Akt2 and Akt3 show elevated mRNA level needs further study on function of Akt isoforms in neuroblastoma.

References

Cheng JQ, Ruggeri B, Klein WM, Sonoda G, Altomare DA, Watson DK, et al. Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA. Proc Natl Acad Sci U S A 1996;93:3636 C41.
Kim CK , Truong L.X. Nguyen, Lee SB, Park SB ,Cho SW , Ahn JY. Lee KH. Akt2 and nucleophosmin/B23 function as an oncogenic unit in human lung cancer cells. Experimental cell research. 317(2011)966 975
Staal SP. Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2:amplification of AKT1 in a primary human gastric adenocarcinoma. Proc Natl Acad Sci U S A. 1987;84:5034 C7.
Stahl JM, Sharma A, Cheung M, Zimmerman M, Cheng JQ, Bosenberg MW, et al. Deregulated Akt3 activity promotes development of malignant melanoma. Cancer Res 2004;64:7002 C10

Figures


Figure 1-Primer locations of Akt1, Akt2 and Akt3


Figure 2-mRNA expression of Akt1, Akt2 and Akt3. RT-PCR was performed in A549, Caco-2 and K562 cell lines. Products are fractionated on 2% agarose gels. GAPDH is used as loading control.


Figure 3-mRNA expression of Akt1, Akt2 and Akt3. RT-PCR was performed in neuroblastoma cell lines:JMN, LAI5-S and LAI-55N. Products are fractionated on 2% agarose gels. GAPDH is used as loading control.


Abstract

The serine/threonine protein kinase Akt (PKB) participates in the PI3K/Akt signal transduction pathway, which plays an important role in the regulation of diverse cellular activities. The Akt family has three highly homologous isoforms: Akt1, Akt2 and Akt3. The three isoforms are encoded by three different genes. Research shows that elevated expression of Akt isoforms are found in different tumors. In this study, mRNA expression of Akt1, Akt2 and Akt3 were detected in six different human cancer cell lines: A549, Caco-2, K562, JMN, LAI-5S and LAI-55N. JMN, LAI-5S and LAI-55N are neuroblastoma cell lines of various malignancies. RT-PCR detected presence of all three Akt isoforms in each cell line studied. Akt2 and Akt3 exhibited differential mRNA expression while no difference in Akt1 mRNA expression was observed.

Full Paper

Acknowledgments

I would like to give my most sincere appreciation to Professor Rubin, who provides useful instructions and guidance in this project. Also, I am thankful to the two TAs, Alex Bulanov and Xie Xie, who did tremendous work to help and support me. Thanks for Professor Ross, who provide the neuroblastoma cell lines for this project.


This document was last modified 05/22/2011.
This site is powered by the versatile Zope platform.
This is a project of the Biology Department of Fordham University
Biotechniques.org Home