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ASPM and microcephalin expression in epithelial ovarian cancer correlates with tumour grade and survival.

Brüning-Richardson A, Bond J, Alsiary R, Richardson J, Cairns DA, McCormack L, Hutson R, Burns P, Wilkinson N, Hall GD, Morrison EE, Bell SM - Br. J. Cancer (2011)

Bottom Line: Two microcephaly-associated proteins, abnormal spindle-like microcephaly-associated protein (ASPM) and microcephalin, are involved in mitosis and DNA damage repair.Their expression is deregulated at the RNA level in EOC.Furthermore, low levels of nuclear microcephalin correlated with reduced patient survival.

View Article: PubMed Central - PubMed

Affiliation: Section of Ophthalmology and Neurosciences, Leeds Institute of Molecular Medicine, Welcome Trust Brenner Building, St James's University Hospital, Leeds LS9 7TF, UK.

ABSTRACT

Background: The clinico-pathological and molecular heterogeneity of epithelial ovarian cancer (EOC) complicates its early diagnosis and successful treatment. Highly aneuploid tumours and the presence of ascitic fluids are hallmarks of EOC. Two microcephaly-associated proteins, abnormal spindle-like microcephaly-associated protein (ASPM) and microcephalin, are involved in mitosis and DNA damage repair. Their expression is deregulated at the RNA level in EOC. Here, ASPM and microcephalin protein expression in primary cultures established from the ascites of patients with EOC was determined and correlated with clinical data to assess their suitability as biomarkers.

Methods: Five established ovarian cancer cell lines, cells derived from two benign ovarian ascites samples and 40 primary cultures of EOC derived from ovarian ascites samples were analysed by protein slot blotting and/or immunofluorescence to determine ASPM and microcephalin protein levels and their cellular localisation. Results were correlated with clinico-pathological data.

Results: A statistically significant correlation was identified for ASPM localisation and tumour grade, with high levels of cytoplasmic ASPM correlating with grade 1 tumours. Conversely, cytoplasmic microcephalin was only identified in high-grade tumours. Furthermore, low levels of nuclear microcephalin correlated with reduced patient survival.

Conclusion: Our results suggest that ASPM and microcephalin have the potential to be biomarkers in ovarian cancer.

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Related in: MedlinePlus

Established ovarian cell lines and primary cultures display different ASPM levels after fluorescence quantification. (A) Histogram illustrating the range of ASPM levels in the nucleus established by immunofluorescence (n=21). Examples of nuclei with low (a, b) and high (c, d) ASPM levels are shown. Green staining indicates ASPM, red α -tubulin and blue DAPI. Scale bars are 5 μm. (B) Histogram illustrating the range of ASPM levels in the cytoplasm as established by immunofluorescence. Examples of low (a, b) and high (c, d) cytoplasmic ASPM levels are shown (n=22). Scale bars are 10 μm (b) and 5 μm (d). (C) Histogram illustrating the range of ASPM levels at the spindle poles as established by immunofluorescence (n=14). Examples of spindle poles with low (a, b) and high (c, d) ASPM levels are shown. Scale bar is 5 μm. Green staining indicates ASPM, red α-tubulin and blue DAPI. (D) Graph depicting an association of cytoplasmic ASPM levels in primary cultures with tumour grade. High cytoplasmic ASPM levels are lost in high-grade tumours (P=0.0351).
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fig3: Established ovarian cell lines and primary cultures display different ASPM levels after fluorescence quantification. (A) Histogram illustrating the range of ASPM levels in the nucleus established by immunofluorescence (n=21). Examples of nuclei with low (a, b) and high (c, d) ASPM levels are shown. Green staining indicates ASPM, red α -tubulin and blue DAPI. Scale bars are 5 μm. (B) Histogram illustrating the range of ASPM levels in the cytoplasm as established by immunofluorescence. Examples of low (a, b) and high (c, d) cytoplasmic ASPM levels are shown (n=22). Scale bars are 10 μm (b) and 5 μm (d). (C) Histogram illustrating the range of ASPM levels at the spindle poles as established by immunofluorescence (n=14). Examples of spindle poles with low (a, b) and high (c, d) ASPM levels are shown. Scale bar is 5 μm. Green staining indicates ASPM, red α-tubulin and blue DAPI. (D) Graph depicting an association of cytoplasmic ASPM levels in primary cultures with tumour grade. High cytoplasmic ASPM levels are lost in high-grade tumours (P=0.0351).

Mentions: Having established that at both the mRNA and protein levels there were differences in ASPM expression among established ovarian cancer cell lines and the primary cultures, we next investigated the cellular localisation and expression levels of ASPM using immunofluorescence. Staining with the ASPM antibody revealed that there was cytoplasmic and nuclear staining in interphase cells in all of the samples as well as spindle pole staining in mitotic cells (Figure 2A). However, the staining patterns and intensity differed among samples and were scored as weak/medium/strong expression at each localisation after quantifying fluorescence intensities. Staining patterns in two samples (1078-A1 and 1153-A1, primary cultures from peritoneal washes from patients with benign gynaecological conditions) revealed weak nuclear and cytoplasmic staining with medium ASPM labelling at the spindle poles (Supplementary Figure 5A). It seems likely that these observations represent typical ASPM localisations and expression levels in ‘normal' control cells. The majority of the 18 tumour-derived samples had both weak nuclear and weak cytoplasmic staining (64.7 and 77.8%, respectively), whereas 50% of the samples had weak mitotic ASPM staining. Medium staining was observed for 23.5% (nuclear), 11.1% (cytoplasmic) and 21.4% (spindle poles) of cultures. Strong nuclear staining was observed in 11.8% of the cultures, strong cytoplasmic staining in 11.1% and strong spindle pole staining in 28.6% (Figure 2B). Analysis of the staining data indicated that the majority of the primary cultures had nuclear- and spindle pole-staining intensities similar to the intensities obtained for the established cell lines. Only two samples (1134-A1 and 1108-A1) for both nuclear staining and cytoplasmic staining and two samples (1140-A1 and 1105-A1) for spindle pole staining had markedly higher levels of ASPM than were observed in the cell lines (Figures 3A–C). Generally, levels of cytoplasmic staining were most variable among the primary cultures (Figure 3B). Statistical analysis for the 18 primary culture samples with associated clinical data indicated that strong cytoplasmic ASPM staining levels were significantly associated with a low tumour grade (P=0.0351; Figure 3D).


ASPM and microcephalin expression in epithelial ovarian cancer correlates with tumour grade and survival.

Brüning-Richardson A, Bond J, Alsiary R, Richardson J, Cairns DA, McCormack L, Hutson R, Burns P, Wilkinson N, Hall GD, Morrison EE, Bell SM - Br. J. Cancer (2011)

Established ovarian cell lines and primary cultures display different ASPM levels after fluorescence quantification. (A) Histogram illustrating the range of ASPM levels in the nucleus established by immunofluorescence (n=21). Examples of nuclei with low (a, b) and high (c, d) ASPM levels are shown. Green staining indicates ASPM, red α -tubulin and blue DAPI. Scale bars are 5 μm. (B) Histogram illustrating the range of ASPM levels in the cytoplasm as established by immunofluorescence. Examples of low (a, b) and high (c, d) cytoplasmic ASPM levels are shown (n=22). Scale bars are 10 μm (b) and 5 μm (d). (C) Histogram illustrating the range of ASPM levels at the spindle poles as established by immunofluorescence (n=14). Examples of spindle poles with low (a, b) and high (c, d) ASPM levels are shown. Scale bar is 5 μm. Green staining indicates ASPM, red α-tubulin and blue DAPI. (D) Graph depicting an association of cytoplasmic ASPM levels in primary cultures with tumour grade. High cytoplasmic ASPM levels are lost in high-grade tumours (P=0.0351).
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Related In: Results  -  Collection

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fig3: Established ovarian cell lines and primary cultures display different ASPM levels after fluorescence quantification. (A) Histogram illustrating the range of ASPM levels in the nucleus established by immunofluorescence (n=21). Examples of nuclei with low (a, b) and high (c, d) ASPM levels are shown. Green staining indicates ASPM, red α -tubulin and blue DAPI. Scale bars are 5 μm. (B) Histogram illustrating the range of ASPM levels in the cytoplasm as established by immunofluorescence. Examples of low (a, b) and high (c, d) cytoplasmic ASPM levels are shown (n=22). Scale bars are 10 μm (b) and 5 μm (d). (C) Histogram illustrating the range of ASPM levels at the spindle poles as established by immunofluorescence (n=14). Examples of spindle poles with low (a, b) and high (c, d) ASPM levels are shown. Scale bar is 5 μm. Green staining indicates ASPM, red α-tubulin and blue DAPI. (D) Graph depicting an association of cytoplasmic ASPM levels in primary cultures with tumour grade. High cytoplasmic ASPM levels are lost in high-grade tumours (P=0.0351).
Mentions: Having established that at both the mRNA and protein levels there were differences in ASPM expression among established ovarian cancer cell lines and the primary cultures, we next investigated the cellular localisation and expression levels of ASPM using immunofluorescence. Staining with the ASPM antibody revealed that there was cytoplasmic and nuclear staining in interphase cells in all of the samples as well as spindle pole staining in mitotic cells (Figure 2A). However, the staining patterns and intensity differed among samples and were scored as weak/medium/strong expression at each localisation after quantifying fluorescence intensities. Staining patterns in two samples (1078-A1 and 1153-A1, primary cultures from peritoneal washes from patients with benign gynaecological conditions) revealed weak nuclear and cytoplasmic staining with medium ASPM labelling at the spindle poles (Supplementary Figure 5A). It seems likely that these observations represent typical ASPM localisations and expression levels in ‘normal' control cells. The majority of the 18 tumour-derived samples had both weak nuclear and weak cytoplasmic staining (64.7 and 77.8%, respectively), whereas 50% of the samples had weak mitotic ASPM staining. Medium staining was observed for 23.5% (nuclear), 11.1% (cytoplasmic) and 21.4% (spindle poles) of cultures. Strong nuclear staining was observed in 11.8% of the cultures, strong cytoplasmic staining in 11.1% and strong spindle pole staining in 28.6% (Figure 2B). Analysis of the staining data indicated that the majority of the primary cultures had nuclear- and spindle pole-staining intensities similar to the intensities obtained for the established cell lines. Only two samples (1134-A1 and 1108-A1) for both nuclear staining and cytoplasmic staining and two samples (1140-A1 and 1105-A1) for spindle pole staining had markedly higher levels of ASPM than were observed in the cell lines (Figures 3A–C). Generally, levels of cytoplasmic staining were most variable among the primary cultures (Figure 3B). Statistical analysis for the 18 primary culture samples with associated clinical data indicated that strong cytoplasmic ASPM staining levels were significantly associated with a low tumour grade (P=0.0351; Figure 3D).

Bottom Line: Two microcephaly-associated proteins, abnormal spindle-like microcephaly-associated protein (ASPM) and microcephalin, are involved in mitosis and DNA damage repair.Their expression is deregulated at the RNA level in EOC.Furthermore, low levels of nuclear microcephalin correlated with reduced patient survival.

View Article: PubMed Central - PubMed

Affiliation: Section of Ophthalmology and Neurosciences, Leeds Institute of Molecular Medicine, Welcome Trust Brenner Building, St James's University Hospital, Leeds LS9 7TF, UK.

ABSTRACT

Background: The clinico-pathological and molecular heterogeneity of epithelial ovarian cancer (EOC) complicates its early diagnosis and successful treatment. Highly aneuploid tumours and the presence of ascitic fluids are hallmarks of EOC. Two microcephaly-associated proteins, abnormal spindle-like microcephaly-associated protein (ASPM) and microcephalin, are involved in mitosis and DNA damage repair. Their expression is deregulated at the RNA level in EOC. Here, ASPM and microcephalin protein expression in primary cultures established from the ascites of patients with EOC was determined and correlated with clinical data to assess their suitability as biomarkers.

Methods: Five established ovarian cancer cell lines, cells derived from two benign ovarian ascites samples and 40 primary cultures of EOC derived from ovarian ascites samples were analysed by protein slot blotting and/or immunofluorescence to determine ASPM and microcephalin protein levels and their cellular localisation. Results were correlated with clinico-pathological data.

Results: A statistically significant correlation was identified for ASPM localisation and tumour grade, with high levels of cytoplasmic ASPM correlating with grade 1 tumours. Conversely, cytoplasmic microcephalin was only identified in high-grade tumours. Furthermore, low levels of nuclear microcephalin correlated with reduced patient survival.

Conclusion: Our results suggest that ASPM and microcephalin have the potential to be biomarkers in ovarian cancer.

Show MeSH
Related in: MedlinePlus