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NUCKS overexpression in breast cancer.

Drosos Y, Kouloukoussa M, Østvold AC, Grundt K, Goutas N, Vlachodimitropoulos D, Havaki S, Kollia P, Kittas C, Marinos E, Aleporou-Marinou V - Cancer Cell Int. (2009)

Bottom Line: This increase was also confirmed by Western immunoblot analysis.Although NUCKS is a cell cycle related protein, its expression does not correlate with Ki67 expression, neither in tissue sections nor in primary cell cultures.In particular, the NUCKS overexpression in ADH and DCIS indicates a significant role of this protein in neoplastic progression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics and Biotechnology, Faculty of Biology, University of Athens, Panepistimioupoli, 15701 Ilissia, Greece. ydrosos@biol.uoa.gr

ABSTRACT

Background: NUCKS (Nuclear, Casein Kinase and Cyclin-dependent Kinase Substrate) is a nuclear, DNA-binding and highly phosphorylated protein. A number of reports show that NUCKS is highly expressed on the level of mRNA in several human cancers, including breast cancer. In this work, NUCKS expression on both RNA and protein levels was studied in breast tissue biopsies consisted of invasive carcinomas, intraductal proliferative lesions, benign epithelial proliferations and fibroadenomas, as well as in primary cultures derived from the above biopsies. Specifically, in order to evaluate the level of NUCKS protein in correlation with the histopathological features of breast disease, immunohistochemistry was employed on paraffin sections of breast biopsies of the above types. In addition, NUCKS expression was studied by means of Reverse Transcription PCR (RT-PCR), real-time PCR (qRT-PCR) and Western immunoblot analyses in the primary cell cultures developed from the same biopsies.

Results: The immunohistochemical Results showed intense NUCKS staining mostly in grade I and II breast carcinomas compared to normal tissues. Furthermore, NUCKS was moderate expressed in benign epithelial proliferations, such as adenosis and sclerosing adenosis, and highly expressed in intraductal lesions, specifically in ductal carcinomas in situ (DCIS). It is worth noting that all the fibroadenoma tissues examined were negative for NUCKS staining. RT-PCR and qRT-PCR showed an increase of NUCKS expression in cells derived from primary cultures of proliferative lesions and cancerous tissues compared to the ones derived from normal breast tissues and fibroadenomas. This increase was also confirmed by Western immunoblot analysis. Although NUCKS is a cell cycle related protein, its expression does not correlate with Ki67 expression, neither in tissue sections nor in primary cell cultures.

Conclusion: The results show overexpression of the NUCKS protein in a number of non malignant breast lesions and cancerous tissues. In particular, the NUCKS overexpression in ADH and DCIS indicates a significant role of this protein in neoplastic progression.

No MeSH data available.


Related in: MedlinePlus

Semi-quantitative mRNA expression of NUCKS in primary cultures from different biopsies. (A) The RT-PCR products, generated with NUCKS and GAPDH gene specific primers, were electrophorized in a 2% agarose gel. GAPDH mRNA was used as an internal control. The most representative cases are illustrated. (B) Graphical presentation of the ratio of NUCKS to GAPDH mRNA levels corresponding to the samples illustrated in (A), as median values of 3 independent experiments (p = 0.05). The mRNA levels were quantitated semiquantitatively as described in the Methods section. TC01, primary culture of normal tissue; TC05, primary culture of fibroadenoma; TC11 and TC13, derived from primary cultures from biopsies with benign epithelial proliferations; TC16, TC20, TC27-TC31 derived from primary cultures from IDC, grade II biopsies; TC32 and TC36 derived from IDC, grade III. The clinicopathological variables of the samples are summarized in Additional file 1. MDA MB-231 and MCF-7 represent cell lines used as a positive control for NUCKS expression. (C) Median values of the ratio of NUCKS to GAPDH mRNA levels in the studied groups (p = 0.05).
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Figure 4: Semi-quantitative mRNA expression of NUCKS in primary cultures from different biopsies. (A) The RT-PCR products, generated with NUCKS and GAPDH gene specific primers, were electrophorized in a 2% agarose gel. GAPDH mRNA was used as an internal control. The most representative cases are illustrated. (B) Graphical presentation of the ratio of NUCKS to GAPDH mRNA levels corresponding to the samples illustrated in (A), as median values of 3 independent experiments (p = 0.05). The mRNA levels were quantitated semiquantitatively as described in the Methods section. TC01, primary culture of normal tissue; TC05, primary culture of fibroadenoma; TC11 and TC13, derived from primary cultures from biopsies with benign epithelial proliferations; TC16, TC20, TC27-TC31 derived from primary cultures from IDC, grade II biopsies; TC32 and TC36 derived from IDC, grade III. The clinicopathological variables of the samples are summarized in Additional file 1. MDA MB-231 and MCF-7 represent cell lines used as a positive control for NUCKS expression. (C) Median values of the ratio of NUCKS to GAPDH mRNA levels in the studied groups (p = 0.05).

Mentions: Using semi-quantitative RT-PCR, the mRNA levels of NUCKS were evaluated in primary culture cells. The clinicopathological parameters of breast biopsies used for primary cultures are summarized in Additional file 1. In Figure 4, the representative results of each clinicopathological category are demonstrated. Specifically, in the primary cultures from normal tissues and fibroadenomas, NUCKS mRNA was detected at low levels. In the cultures derived from tissues with benign lesions, NUCKS mRNA varied from low levels, as observed in normal tissues and fibroadenomas, to higher levels resembling some cases of IDC, grade II. In most of the cultures derived from grade II tumors, NUCKS mRNA was significantly increased compared to normal. However, there were some cultures from grade II carcinomas exhibiting low NUCKS mRNA levels. The observed variation of NUCKS mRNA in primary cultures from benign and grade II biopsies is concomitant to immunohistochemical results from the corresponding biopsies (Tables 1 and 2) and may be related to tumor heterogeneity. In most of the cultures derived from biopsies of IDC, grade III, NUCKS mRNA levels were lower than that observed in primary cultures from IDC, grade II, while in few cases the levels of NUCKS mRNA expression were significantly higher.


NUCKS overexpression in breast cancer.

Drosos Y, Kouloukoussa M, Østvold AC, Grundt K, Goutas N, Vlachodimitropoulos D, Havaki S, Kollia P, Kittas C, Marinos E, Aleporou-Marinou V - Cancer Cell Int. (2009)

Semi-quantitative mRNA expression of NUCKS in primary cultures from different biopsies. (A) The RT-PCR products, generated with NUCKS and GAPDH gene specific primers, were electrophorized in a 2% agarose gel. GAPDH mRNA was used as an internal control. The most representative cases are illustrated. (B) Graphical presentation of the ratio of NUCKS to GAPDH mRNA levels corresponding to the samples illustrated in (A), as median values of 3 independent experiments (p = 0.05). The mRNA levels were quantitated semiquantitatively as described in the Methods section. TC01, primary culture of normal tissue; TC05, primary culture of fibroadenoma; TC11 and TC13, derived from primary cultures from biopsies with benign epithelial proliferations; TC16, TC20, TC27-TC31 derived from primary cultures from IDC, grade II biopsies; TC32 and TC36 derived from IDC, grade III. The clinicopathological variables of the samples are summarized in Additional file 1. MDA MB-231 and MCF-7 represent cell lines used as a positive control for NUCKS expression. (C) Median values of the ratio of NUCKS to GAPDH mRNA levels in the studied groups (p = 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2743642&req=5

Figure 4: Semi-quantitative mRNA expression of NUCKS in primary cultures from different biopsies. (A) The RT-PCR products, generated with NUCKS and GAPDH gene specific primers, were electrophorized in a 2% agarose gel. GAPDH mRNA was used as an internal control. The most representative cases are illustrated. (B) Graphical presentation of the ratio of NUCKS to GAPDH mRNA levels corresponding to the samples illustrated in (A), as median values of 3 independent experiments (p = 0.05). The mRNA levels were quantitated semiquantitatively as described in the Methods section. TC01, primary culture of normal tissue; TC05, primary culture of fibroadenoma; TC11 and TC13, derived from primary cultures from biopsies with benign epithelial proliferations; TC16, TC20, TC27-TC31 derived from primary cultures from IDC, grade II biopsies; TC32 and TC36 derived from IDC, grade III. The clinicopathological variables of the samples are summarized in Additional file 1. MDA MB-231 and MCF-7 represent cell lines used as a positive control for NUCKS expression. (C) Median values of the ratio of NUCKS to GAPDH mRNA levels in the studied groups (p = 0.05).
Mentions: Using semi-quantitative RT-PCR, the mRNA levels of NUCKS were evaluated in primary culture cells. The clinicopathological parameters of breast biopsies used for primary cultures are summarized in Additional file 1. In Figure 4, the representative results of each clinicopathological category are demonstrated. Specifically, in the primary cultures from normal tissues and fibroadenomas, NUCKS mRNA was detected at low levels. In the cultures derived from tissues with benign lesions, NUCKS mRNA varied from low levels, as observed in normal tissues and fibroadenomas, to higher levels resembling some cases of IDC, grade II. In most of the cultures derived from grade II tumors, NUCKS mRNA was significantly increased compared to normal. However, there were some cultures from grade II carcinomas exhibiting low NUCKS mRNA levels. The observed variation of NUCKS mRNA in primary cultures from benign and grade II biopsies is concomitant to immunohistochemical results from the corresponding biopsies (Tables 1 and 2) and may be related to tumor heterogeneity. In most of the cultures derived from biopsies of IDC, grade III, NUCKS mRNA levels were lower than that observed in primary cultures from IDC, grade II, while in few cases the levels of NUCKS mRNA expression were significantly higher.

Bottom Line: This increase was also confirmed by Western immunoblot analysis.Although NUCKS is a cell cycle related protein, its expression does not correlate with Ki67 expression, neither in tissue sections nor in primary cell cultures.In particular, the NUCKS overexpression in ADH and DCIS indicates a significant role of this protein in neoplastic progression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics and Biotechnology, Faculty of Biology, University of Athens, Panepistimioupoli, 15701 Ilissia, Greece. ydrosos@biol.uoa.gr

ABSTRACT

Background: NUCKS (Nuclear, Casein Kinase and Cyclin-dependent Kinase Substrate) is a nuclear, DNA-binding and highly phosphorylated protein. A number of reports show that NUCKS is highly expressed on the level of mRNA in several human cancers, including breast cancer. In this work, NUCKS expression on both RNA and protein levels was studied in breast tissue biopsies consisted of invasive carcinomas, intraductal proliferative lesions, benign epithelial proliferations and fibroadenomas, as well as in primary cultures derived from the above biopsies. Specifically, in order to evaluate the level of NUCKS protein in correlation with the histopathological features of breast disease, immunohistochemistry was employed on paraffin sections of breast biopsies of the above types. In addition, NUCKS expression was studied by means of Reverse Transcription PCR (RT-PCR), real-time PCR (qRT-PCR) and Western immunoblot analyses in the primary cell cultures developed from the same biopsies.

Results: The immunohistochemical Results showed intense NUCKS staining mostly in grade I and II breast carcinomas compared to normal tissues. Furthermore, NUCKS was moderate expressed in benign epithelial proliferations, such as adenosis and sclerosing adenosis, and highly expressed in intraductal lesions, specifically in ductal carcinomas in situ (DCIS). It is worth noting that all the fibroadenoma tissues examined were negative for NUCKS staining. RT-PCR and qRT-PCR showed an increase of NUCKS expression in cells derived from primary cultures of proliferative lesions and cancerous tissues compared to the ones derived from normal breast tissues and fibroadenomas. This increase was also confirmed by Western immunoblot analysis. Although NUCKS is a cell cycle related protein, its expression does not correlate with Ki67 expression, neither in tissue sections nor in primary cell cultures.

Conclusion: The results show overexpression of the NUCKS protein in a number of non malignant breast lesions and cancerous tissues. In particular, the NUCKS overexpression in ADH and DCIS indicates a significant role of this protein in neoplastic progression.

No MeSH data available.


Related in: MedlinePlus