Limits...
Tetratricopeptide repeat domain 9A is an interacting protein for tropomyosin Tm5NM-1.

Cao S, Ho GH, Lin VC - BMC Cancer (2008)

Bottom Line: Tropomyosin Tm5NM-1 was identified as one of the TTC9A partner proteins.The results suggested that the first TPR domain and the linker fragment between the first two TPR domains of TTC9A were important for the interaction with Tm5NM-1 and the second and the third TPR might play an inhibitory role.In our previous results, we have found that progesterone-induced TTC9A expression was associated with increased cell motility and cell spreading.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore. ashleycsl@gmail.com

ABSTRACT

Background: Tetratricopeptide repeat domain 9A (TTC9A) protein is a recently identified protein which contains three tetratricopeptide repeats (TPRs) on its C-terminus. In our previous studies, we have shown that TTC9A was a hormonally-regulated gene in breast cancer cells. In this study, we found that TTC9A was over-expressed in breast cancer tissues compared with the adjacent controls (P < 0.00001), suggesting it might be involved in the breast cancer development process. The aim of the current study was to further elucidate the function of TTC9A.

Methods: Breast samples from 25 patients including the malignant breast tissues and the adjacent normal tissues were processed for Southern blot analysis. Yeast-two-hybrid assay, GST pull-down assay and co-immunoprecipitation were used to identify and verify the interaction between TTC9A and other proteins.

Results: Tropomyosin Tm5NM-1 was identified as one of the TTC9A partner proteins. The interaction between TTC9A and Tm5NM-1 was further confirmed by GST pull-down assay and co-immunoprecipitation in mammalian cells. TTC9A domains required for the interaction were also characterized in this study. The results suggested that the first TPR domain and the linker fragment between the first two TPR domains of TTC9A were important for the interaction with Tm5NM-1 and the second and the third TPR might play an inhibitory role.

Conclusion: Since the primary function of tropomyosin is to stabilize actin filament, its interaction with TTC9A may play a role in cell shape and motility. In our previous results, we have found that progesterone-induced TTC9A expression was associated with increased cell motility and cell spreading. We speculate that TTC9A acts as a chaperone protein to facilitate the function of tropomyosins in stabilizing microfilament and it may play a role in cancer cell invasion and metastasis.

Show MeSH

Related in: MedlinePlus

The expression level of TTC9A mRNA was significantly higher in breast cancer tissues than that in the adjacent normal breast tissues. Total RNA was extracted from human breast cancer tissues and the matched adjacent normal breast tissues. Equal amount of RNA from each sample was subjected to reverse transcription and cDNA produced was amplified by PCR using TTC9A, 36B4 or GAPDH primers. 10 μl PCR products were separated on an agarose gel and analyzed by Southern blotting. Band intensity was analyzed by Bio-Rad Molecular Image Analyzer. The figure shows the expression levels of TTC9A in 25 pairs of normal and tumor tissue samples after normalizing to those of 36B4 (A) or GAPDH (B). Each pair of bars represents samples from one patient. The primers used for TTC9A were 5'-CACATGTCTATAACGATTT CC-3' (forward) and 5'-TGCAGGAAACAGGGG ACTCTC-3' (reverse). The primers used to amplify 36B4 gene were 5'-GATTGGCTACCCAACTGTTGCA-3' (forward) and 5'-CAGGGGCAGCAGCCACAAAGGC-3' (reverse). The primers for GAPDH were 5'-TGCACCACCAACTGCTTAG-3' (forward) and 5'-GAGGCAGGGATGATG TTC-3' (reverse).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2538545&req=5

Figure 1: The expression level of TTC9A mRNA was significantly higher in breast cancer tissues than that in the adjacent normal breast tissues. Total RNA was extracted from human breast cancer tissues and the matched adjacent normal breast tissues. Equal amount of RNA from each sample was subjected to reverse transcription and cDNA produced was amplified by PCR using TTC9A, 36B4 or GAPDH primers. 10 μl PCR products were separated on an agarose gel and analyzed by Southern blotting. Band intensity was analyzed by Bio-Rad Molecular Image Analyzer. The figure shows the expression levels of TTC9A in 25 pairs of normal and tumor tissue samples after normalizing to those of 36B4 (A) or GAPDH (B). Each pair of bars represents samples from one patient. The primers used for TTC9A were 5'-CACATGTCTATAACGATTT CC-3' (forward) and 5'-TGCAGGAAACAGGGG ACTCTC-3' (reverse). The primers used to amplify 36B4 gene were 5'-GATTGGCTACCCAACTGTTGCA-3' (forward) and 5'-CAGGGGCAGCAGCCACAAAGGC-3' (reverse). The primers for GAPDH were 5'-TGCACCACCAACTGCTTAG-3' (forward) and 5'-GAGGCAGGGATGATG TTC-3' (reverse).

Mentions: Previous results have shown that TTC9A is a hormonally regulated gene in vitro [2]. As breast cancer is well-known to be a hormone-dependent malignancy, it is noteworthy to know whether TTC9A is over-expressed in breast cancer tissues and if its expression is correlated with hormone receptor status. 25 matched pairs of human breast cancer tissue and the adjacent normal tissue were analyzed for TTC9A mRNA expression. The results presented in Fig. 1 were obtained by RT-PCR as illustrated in the "materials and methods" and the PCR products were quantitated by Southern blotting analysis. 36B4, which codes for human acidic ribosomal phosphoprotein P0, was used as a control for cDNA input (Fig. 1A). Since the expression of 36B4 varied among samples, GAPDH was also included as a normalization standard for cDNA input. It turned out that the expression levels of these housekeeping genes were always higher in tumor tissues compared with the corresponding normal tissues. This variation has been reported before. For example, GAPDH expression was 3.3-fold higher in seminoma compared with normal testis [38]. Similarly, GAPDH transcription was significantly greater in both colonic adenomas and cancers than in normal mucosa [39]. Nonetheless, the results revealed that the relative expression of TTC9A mRNA, when normalized to either 36B4 or GAPDH, was significantly higher in breast cancer tissue compared with its adjacent normal tissue (Fig. 1) (P < 0.00001). It is also notable that nearly all the tumor tissues expressed higher level of TTC9A mRNA compared with its adjacent controls. However, we found no correlation of TTC9A expression with other clinic pathological data such as tumor size, nuclear grade, axillary's lymph node status or hormone receptor expression.


Tetratricopeptide repeat domain 9A is an interacting protein for tropomyosin Tm5NM-1.

Cao S, Ho GH, Lin VC - BMC Cancer (2008)

The expression level of TTC9A mRNA was significantly higher in breast cancer tissues than that in the adjacent normal breast tissues. Total RNA was extracted from human breast cancer tissues and the matched adjacent normal breast tissues. Equal amount of RNA from each sample was subjected to reverse transcription and cDNA produced was amplified by PCR using TTC9A, 36B4 or GAPDH primers. 10 μl PCR products were separated on an agarose gel and analyzed by Southern blotting. Band intensity was analyzed by Bio-Rad Molecular Image Analyzer. The figure shows the expression levels of TTC9A in 25 pairs of normal and tumor tissue samples after normalizing to those of 36B4 (A) or GAPDH (B). Each pair of bars represents samples from one patient. The primers used for TTC9A were 5'-CACATGTCTATAACGATTT CC-3' (forward) and 5'-TGCAGGAAACAGGGG ACTCTC-3' (reverse). The primers used to amplify 36B4 gene were 5'-GATTGGCTACCCAACTGTTGCA-3' (forward) and 5'-CAGGGGCAGCAGCCACAAAGGC-3' (reverse). The primers for GAPDH were 5'-TGCACCACCAACTGCTTAG-3' (forward) and 5'-GAGGCAGGGATGATG TTC-3' (reverse).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2538545&req=5

Figure 1: The expression level of TTC9A mRNA was significantly higher in breast cancer tissues than that in the adjacent normal breast tissues. Total RNA was extracted from human breast cancer tissues and the matched adjacent normal breast tissues. Equal amount of RNA from each sample was subjected to reverse transcription and cDNA produced was amplified by PCR using TTC9A, 36B4 or GAPDH primers. 10 μl PCR products were separated on an agarose gel and analyzed by Southern blotting. Band intensity was analyzed by Bio-Rad Molecular Image Analyzer. The figure shows the expression levels of TTC9A in 25 pairs of normal and tumor tissue samples after normalizing to those of 36B4 (A) or GAPDH (B). Each pair of bars represents samples from one patient. The primers used for TTC9A were 5'-CACATGTCTATAACGATTT CC-3' (forward) and 5'-TGCAGGAAACAGGGG ACTCTC-3' (reverse). The primers used to amplify 36B4 gene were 5'-GATTGGCTACCCAACTGTTGCA-3' (forward) and 5'-CAGGGGCAGCAGCCACAAAGGC-3' (reverse). The primers for GAPDH were 5'-TGCACCACCAACTGCTTAG-3' (forward) and 5'-GAGGCAGGGATGATG TTC-3' (reverse).
Mentions: Previous results have shown that TTC9A is a hormonally regulated gene in vitro [2]. As breast cancer is well-known to be a hormone-dependent malignancy, it is noteworthy to know whether TTC9A is over-expressed in breast cancer tissues and if its expression is correlated with hormone receptor status. 25 matched pairs of human breast cancer tissue and the adjacent normal tissue were analyzed for TTC9A mRNA expression. The results presented in Fig. 1 were obtained by RT-PCR as illustrated in the "materials and methods" and the PCR products were quantitated by Southern blotting analysis. 36B4, which codes for human acidic ribosomal phosphoprotein P0, was used as a control for cDNA input (Fig. 1A). Since the expression of 36B4 varied among samples, GAPDH was also included as a normalization standard for cDNA input. It turned out that the expression levels of these housekeeping genes were always higher in tumor tissues compared with the corresponding normal tissues. This variation has been reported before. For example, GAPDH expression was 3.3-fold higher in seminoma compared with normal testis [38]. Similarly, GAPDH transcription was significantly greater in both colonic adenomas and cancers than in normal mucosa [39]. Nonetheless, the results revealed that the relative expression of TTC9A mRNA, when normalized to either 36B4 or GAPDH, was significantly higher in breast cancer tissue compared with its adjacent normal tissue (Fig. 1) (P < 0.00001). It is also notable that nearly all the tumor tissues expressed higher level of TTC9A mRNA compared with its adjacent controls. However, we found no correlation of TTC9A expression with other clinic pathological data such as tumor size, nuclear grade, axillary's lymph node status or hormone receptor expression.

Bottom Line: Tropomyosin Tm5NM-1 was identified as one of the TTC9A partner proteins.The results suggested that the first TPR domain and the linker fragment between the first two TPR domains of TTC9A were important for the interaction with Tm5NM-1 and the second and the third TPR might play an inhibitory role.In our previous results, we have found that progesterone-induced TTC9A expression was associated with increased cell motility and cell spreading.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore. ashleycsl@gmail.com

ABSTRACT

Background: Tetratricopeptide repeat domain 9A (TTC9A) protein is a recently identified protein which contains three tetratricopeptide repeats (TPRs) on its C-terminus. In our previous studies, we have shown that TTC9A was a hormonally-regulated gene in breast cancer cells. In this study, we found that TTC9A was over-expressed in breast cancer tissues compared with the adjacent controls (P < 0.00001), suggesting it might be involved in the breast cancer development process. The aim of the current study was to further elucidate the function of TTC9A.

Methods: Breast samples from 25 patients including the malignant breast tissues and the adjacent normal tissues were processed for Southern blot analysis. Yeast-two-hybrid assay, GST pull-down assay and co-immunoprecipitation were used to identify and verify the interaction between TTC9A and other proteins.

Results: Tropomyosin Tm5NM-1 was identified as one of the TTC9A partner proteins. The interaction between TTC9A and Tm5NM-1 was further confirmed by GST pull-down assay and co-immunoprecipitation in mammalian cells. TTC9A domains required for the interaction were also characterized in this study. The results suggested that the first TPR domain and the linker fragment between the first two TPR domains of TTC9A were important for the interaction with Tm5NM-1 and the second and the third TPR might play an inhibitory role.

Conclusion: Since the primary function of tropomyosin is to stabilize actin filament, its interaction with TTC9A may play a role in cell shape and motility. In our previous results, we have found that progesterone-induced TTC9A expression was associated with increased cell motility and cell spreading. We speculate that TTC9A acts as a chaperone protein to facilitate the function of tropomyosins in stabilizing microfilament and it may play a role in cancer cell invasion and metastasis.

Show MeSH
Related in: MedlinePlus