Limits...
Independent overexpression of the subunits of translation elongation factor complex eEF1H in human lung cancer.

Veremieva M, Kapustian L, Khoruzhenko A, Zakharychev V, Negrutskii B, El'skaya A - BMC Cancer (2014)

Bottom Line: The components of translation elongation complex eEF1H (eEF1A, eEF1Bα, eEF1Bβ, eEF1Bγ) were found overexpressed in different cancers.Nuclear localization of eEF1Bβ in the cancer rather than distal normal looking tissues was found.In cancer tissue, eEF1A and eEF1Bα were not found in nuclei while all four subunits of eEF1H demonstrated both cytoplasmic and nuclear appearance in the lung carcinoma cell line A549.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Molecular and Cellular Biology, Institute of Molecular Biology and Genetics NASU, 150 Acad,Zabolotnogo Str,, Kiev 03680, Ukraine. negrutskii@imbg.org.ua.

ABSTRACT

Background: The constituents of stable multiprotein complexes are known to dissociate from the complex to play independent regulatory roles. The components of translation elongation complex eEF1H (eEF1A, eEF1Bα, eEF1Bβ, eEF1Bγ) were found overexpressed in different cancers. To gain the knowledge about novel cancer-related translational mechanisms we intended to reveal whether eEF1H exists as a single unit or independent subunits in different human cancers.

Methods: The changes in the expression level of every subunit of eEF1H in the human non-small-cell lung cancer tissues were examined. The localization of eEF1H subunits was assessed by immunohistochemistry methods, subcellular fractionation and confocal microscopy. The possibility of the interaction between the subunits was estimated by co-immunoprecipitation.

Results: The level of eEF1Bβ expression was increased more than two-fold in 36%, eEF1Bγ in 28%, eEF1A in 20% and eEF1Bα in 8% of tumor specimens. The cancer-induced alterations in the subunits level were found to be uncoordinated, therefore the increase in the level of at least one subunit of eEF1H was observed in 52% of samples. Nuclear localization of eEF1Bβ in the cancer rather than distal normal looking tissues was found. In cancer tissue, eEF1A and eEF1Bα were not found in nuclei while all four subunits of eEF1H demonstrated both cytoplasmic and nuclear appearance in the lung carcinoma cell line A549. Unexpectedly, in the A549 nuclear fraction eEF1A lost the ability to interact with the eEF1B complex.

Conclusions: The results suggest independent functioning of some fraction of the eEF1H subunits in human tumors. The absence of eEF1A and eEF1B interplay in nuclei of A549 cells is a first evidence for non-translational role of nuclear-localized subunits of eEF1B. We conclude the appearance of the individual eEF1B subunits in tumors is a more general phenomenon than appreciated before and thus is a novel signal of cancer-related changes in translation apparatus.

Show MeSH

Related in: MedlinePlus

Distribution of the eEF1H subunits in A549 cells.(A) Subcellular fractionation. CE - cytoplasmic extract, NM – nuclear membrane fraction, NE – nuclear extract, C — control (the pellet after taking out the cytoplasmic fraction and before isolation of nuclei). (B) Immunofluorescent analysis of eEF1Bγ localization. (a) Localization of eEF1Bγ in A549 cells, (b) nuclear staining with Hoechst 33342, (c) merge. Scale bar 20 μm. (C) Co-immunoprecipitation of eEF1A and eEF1Bγ. CE – cytoplasmic extract, NE – nuclear extract. IP – fraction of immunoprecipitated proteins, B – fraction of non-bound proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4265501&req=5

Fig4: Distribution of the eEF1H subunits in A549 cells.(A) Subcellular fractionation. CE - cytoplasmic extract, NM – nuclear membrane fraction, NE – nuclear extract, C — control (the pellet after taking out the cytoplasmic fraction and before isolation of nuclei). (B) Immunofluorescent analysis of eEF1Bγ localization. (a) Localization of eEF1Bγ in A549 cells, (b) nuclear staining with Hoechst 33342, (c) merge. Scale bar 20 μm. (C) Co-immunoprecipitation of eEF1A and eEF1Bγ. CE – cytoplasmic extract, NE – nuclear extract. IP – fraction of immunoprecipitated proteins, B – fraction of non-bound proteins.

Mentions: To analyze nuclear localization of the eEF1Bβ and eEF1Bγ subunits in more detail, we isolated the fraction of nuclear proteins from the human lung adenocarcinoma cell line A549. Unexpectedly, all eEF1H subunits were found to be present in both cytoplasmic and nuclear fractions of A549 cells (Figure 4A) which contradicted the data on the cancer tissues where eEF1A and eEF1Bα were not found in nucleus. The nucleus-located eEF1A and eEF1Bα did not seem to be contaminants from outer side of nuclear membrane, as these subunits were not present in the nuclear membrane fraction of A549 cells (Figure 4A). eEF1Bγ was found in all subcellular fractions including the nuclear membrane fraction, indicating the possibility of cross-contamination of the fractions with eEF1Bγ. Therefore, a confocal microscopy of A549 cells was conducted to specify intracellular localization of eEF1Bγ. As seen in Figure 4B, the eEF1Bγ subunit was present in both cytoplasm and nucleus of A549 cells. Thus, in the lung cancer cell line all subunits of eEF1B demonstrate both cytoplasmic and nuclear localization.Figure 4


Independent overexpression of the subunits of translation elongation factor complex eEF1H in human lung cancer.

Veremieva M, Kapustian L, Khoruzhenko A, Zakharychev V, Negrutskii B, El'skaya A - BMC Cancer (2014)

Distribution of the eEF1H subunits in A549 cells.(A) Subcellular fractionation. CE - cytoplasmic extract, NM – nuclear membrane fraction, NE – nuclear extract, C — control (the pellet after taking out the cytoplasmic fraction and before isolation of nuclei). (B) Immunofluorescent analysis of eEF1Bγ localization. (a) Localization of eEF1Bγ in A549 cells, (b) nuclear staining with Hoechst 33342, (c) merge. Scale bar 20 μm. (C) Co-immunoprecipitation of eEF1A and eEF1Bγ. CE – cytoplasmic extract, NE – nuclear extract. IP – fraction of immunoprecipitated proteins, B – fraction of non-bound proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4265501&req=5

Fig4: Distribution of the eEF1H subunits in A549 cells.(A) Subcellular fractionation. CE - cytoplasmic extract, NM – nuclear membrane fraction, NE – nuclear extract, C — control (the pellet after taking out the cytoplasmic fraction and before isolation of nuclei). (B) Immunofluorescent analysis of eEF1Bγ localization. (a) Localization of eEF1Bγ in A549 cells, (b) nuclear staining with Hoechst 33342, (c) merge. Scale bar 20 μm. (C) Co-immunoprecipitation of eEF1A and eEF1Bγ. CE – cytoplasmic extract, NE – nuclear extract. IP – fraction of immunoprecipitated proteins, B – fraction of non-bound proteins.
Mentions: To analyze nuclear localization of the eEF1Bβ and eEF1Bγ subunits in more detail, we isolated the fraction of nuclear proteins from the human lung adenocarcinoma cell line A549. Unexpectedly, all eEF1H subunits were found to be present in both cytoplasmic and nuclear fractions of A549 cells (Figure 4A) which contradicted the data on the cancer tissues where eEF1A and eEF1Bα were not found in nucleus. The nucleus-located eEF1A and eEF1Bα did not seem to be contaminants from outer side of nuclear membrane, as these subunits were not present in the nuclear membrane fraction of A549 cells (Figure 4A). eEF1Bγ was found in all subcellular fractions including the nuclear membrane fraction, indicating the possibility of cross-contamination of the fractions with eEF1Bγ. Therefore, a confocal microscopy of A549 cells was conducted to specify intracellular localization of eEF1Bγ. As seen in Figure 4B, the eEF1Bγ subunit was present in both cytoplasm and nucleus of A549 cells. Thus, in the lung cancer cell line all subunits of eEF1B demonstrate both cytoplasmic and nuclear localization.Figure 4

Bottom Line: The components of translation elongation complex eEF1H (eEF1A, eEF1Bα, eEF1Bβ, eEF1Bγ) were found overexpressed in different cancers.Nuclear localization of eEF1Bβ in the cancer rather than distal normal looking tissues was found.In cancer tissue, eEF1A and eEF1Bα were not found in nuclei while all four subunits of eEF1H demonstrated both cytoplasmic and nuclear appearance in the lung carcinoma cell line A549.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Molecular and Cellular Biology, Institute of Molecular Biology and Genetics NASU, 150 Acad,Zabolotnogo Str,, Kiev 03680, Ukraine. negrutskii@imbg.org.ua.

ABSTRACT

Background: The constituents of stable multiprotein complexes are known to dissociate from the complex to play independent regulatory roles. The components of translation elongation complex eEF1H (eEF1A, eEF1Bα, eEF1Bβ, eEF1Bγ) were found overexpressed in different cancers. To gain the knowledge about novel cancer-related translational mechanisms we intended to reveal whether eEF1H exists as a single unit or independent subunits in different human cancers.

Methods: The changes in the expression level of every subunit of eEF1H in the human non-small-cell lung cancer tissues were examined. The localization of eEF1H subunits was assessed by immunohistochemistry methods, subcellular fractionation and confocal microscopy. The possibility of the interaction between the subunits was estimated by co-immunoprecipitation.

Results: The level of eEF1Bβ expression was increased more than two-fold in 36%, eEF1Bγ in 28%, eEF1A in 20% and eEF1Bα in 8% of tumor specimens. The cancer-induced alterations in the subunits level were found to be uncoordinated, therefore the increase in the level of at least one subunit of eEF1H was observed in 52% of samples. Nuclear localization of eEF1Bβ in the cancer rather than distal normal looking tissues was found. In cancer tissue, eEF1A and eEF1Bα were not found in nuclei while all four subunits of eEF1H demonstrated both cytoplasmic and nuclear appearance in the lung carcinoma cell line A549. Unexpectedly, in the A549 nuclear fraction eEF1A lost the ability to interact with the eEF1B complex.

Conclusions: The results suggest independent functioning of some fraction of the eEF1H subunits in human tumors. The absence of eEF1A and eEF1B interplay in nuclei of A549 cells is a first evidence for non-translational role of nuclear-localized subunits of eEF1B. We conclude the appearance of the individual eEF1B subunits in tumors is a more general phenomenon than appreciated before and thus is a novel signal of cancer-related changes in translation apparatus.

Show MeSH
Related in: MedlinePlus