Expression of γ-tubulin in non-small cell lung cancer and effect on patient survival.

INTRODUCTION: It has been reported that overexpression and altered compartmentalization of γ-tubulin may contribute to tumorigenesis and tumor aggressiveness in a variety of human malignancies. We have shown that γ-tubulin expression and cellular distribution pattern is also altered in non-small cell lung cancer (NSCLC) (Histol. Histopathol. 2012; 27: 1183-1194). In the present study we examined the relationship between γ-tubulin expression and patient overall survival (OS). MATERIAL AND METHODS: Immunohistochemistry was performed, with well-characterized anti-γ-tubulin antibodies, on 109 formalin-fixed, paraffin-embedded NSCLC specimens (p-TNM stage I-III). γ-Tubulin labeling indexes (LIs) were determined, and the association of γ-tubulin expression with clinicopathological parameters was evaluated. To analyze OS rates according to γ-tubulin LIs, patients were categorized into three groups: those with low (0-30%), intermediate (31-69%) or high (70-100%) γ-tubulin LI. Association of clinicopathological parameters and γ-tubulin with survival were examined using univariate and multivariate Cox regression analysis. RESULTS: No statistically significant association was seen between γ-tubulin overexpression and histological type, tumor differentiation, p-TNM stage and adenocarcinoma subtyping. Longer survival was observed in the high γ-tubulin LI group of patients with p-TNM stages II+III when compared to intermediate or low γ-tubulin LI groups, but the difference was not statistically significant (p=0.066). On the other hand, when combined low and intermediate γ-tubulin LI groups (p-TNM stages II+III) where compared to high γ-tubulin LI group, statistically significant longer survival was observed in high γ-tubulin group (p=0.021). CONCLUSION: Our findings suggest that level of γ-tubulin expression may have an impact on patient survival at more advanced NSCLC stages.

Overexpression and Nucleolar Localization of γ-Tubulin Small Complex Proteins GCP2 and GCP3 in Glioblastoma.

The expression, cellular distribution, and subcellular sorting of the microtubule (MT)-nucleating γ-tubulin small complex (γTuSC) proteins, GCP2 and GCP3, were studied in human glioblastoma cell lines and in clinical tissue samples representing all histologic grades of adult diffuse astrocytic gliomas (n = 54). Quantitative real-time polymerase chain reaction revealed a significant increase in the expression of GCP2 and GCP3 transcripts in glioblastoma cells versus normal human astrocytes; these were associated with higher amounts of both γTuSC proteins. GCP2 and GCP3 were concentrated in the centrosomes in interphase glioblastoma cells, but punctate and diffuse localizations were also detected in the cytosol and nuclei/nucleoli. Nucleolar localization was fixation dependent. GCP2 and GCP3 formed complexes with γ-tubulin in the nucleoli as confirmed by reciprocal immunoprecipitation experiments and immunoelectron microscopy. GCP2 and GCP3 depletion caused accumulation of cells in G2/M and mitotic delay but did not affect nucleolar integrity. Overexpression of GCP2 antagonized the inhibitory effect of the CDK5 regulatory subunit-associated tumor suppressor protein 3 (C53) on DNA damage G2/M checkpoint activity. Tumor cell GCP2 and GCP3 immunoreactivity was significantly increased over that in normal brains in glioblastoma samples; it was also associated with microvascular proliferation. These findings suggest that γTuSC protein dysregulation in glioblastomas may be linked to altered transcriptional checkpoint activity or interaction with signaling pathways associated with a malignant phenotype.

Overexpression of γ-tubulin in non-small cell lung cancer.

We and others have previously shown that increased expression and altered compartmentalization of γ-tubulin may contribute to tumorigenesis and tumor progression (J. Cell Physiol. 2009;223:519-529; Cancer Biol. Ther. 2010;9:66-76). Here we have determined by immunohistochemistry the localization and cellular distribution of γ-tubulin in clinical tissue samples from 109 non-small cell lung cancer (NSCLC) cases. The expression and distribution of γ-tubulin protein and transcripts was also determined in the NSCLC tumor cell lines NCI-H460 (HTB-177) and NCI-H69 (HTB-119) by immunocytochemistry, quantitative immunoblotting and reverse transcription quantitative real-time PCR (RT-qPCR). Polyclonal and monoclonal anti-peptide antibodies recognizing epitopes in the C- or N-terminal domains of γ-tubulins and human gene-specific primers for γ-tubulins 1 (TUBG1) and 2 (TUBG2) were used. In non-neoplastic cells of the airway epithelium in situ, γ-tubulin exhibited predominantly apical surface and pericentriolar localizations. In contrast, markedly increased, albeit heterogeneous and variously prominent γ-tubulin immunoreactivity was detected in clinical tumor specimens and in the NCI-H460 and NCI-H69 cell lines, where tumor cells exhibited overlapping multi-punctate and diffuse patterns of localization. Co-expression of γ-tubulin and Ki-67 (MIB-1) was detected in a population of proliferating tumor cells. A statistically significant increase of γ-tubulin expression was found in Stage III compared to lesser stage tumors (p<0.001 v. Stages I/II) regardless of histological subtype or grade. By quantitative immunoblotting NCI-H460 and NCI-H69 cells expressed higher levels of γ-tubulin protein compared to small airway epithelial cells (SAEC). In both tumor cell lines increase in TUBG1 and TUBG2 transcripts was detected by RT-qPCR. Our results reveal for the first time an increased expression of γ-tubulin in lung cancer.

Prognostic impact of Deoxyribonucleic acid (DNA) image analysis cytometry and immunohistochemical expression of Ki67 in surgically resected non-small cell lung cancers.

BACKGROUND: The aim of the present study was to evaluate the prognostic significance of DNA ploidy and Ki67 expression in non-small cell lung carcinoma (NSCLC). METHODS: This prospective study included 96 patients with stages I-IIIA NSCLC who underwent surgical excision. DNA image analysis cytometry was applied on imprints. Calculation of the DNA index (DI) and the 5c exceeding rate (5cER) was performed and the histograms were classified as peridiploid, peritetraploid, and x-ploid-multiploid. The Ki67 immunoreactivity was determined according to the avidin-biotin complex immunoperoxidase method. RESULTS: DNA histogram classification disclosed 30 peridiploid cases, 15 peritetraploid and 51 x-ploid-multiploid. Forty-eight cases (50%) had 5cER > 5%. The Ki67 immunoreactivity was below 25% in 53 tumors (62.4%) and above 25% in 32 (32.6%). Our results revealed the existence of a statistically significant relationship of DNA ploidy with nodal status (p = 0.042) and grade (p = 0.005). Adenocarcinomas and large cell carcinomas were more frequently encountered in x-ploid-multiploid tumors as compared to squamous cell carcinomas, which were more frequently peridiploid (p = 0.003). 5cER showed statistically significant association with nodal status (p = 0.037). Univariate analysis with respect to survival revealed significant association with stage (p < 0.001), nodal status (p < 0.001), tumor status (p < 0.001), DNA ploidy (p = 0.008) and 5cER (p = 0.0124). Multivariate analysis revealed stage and ploidy status as independent factors: peridiploid tumors were associated with better survival as compared to x-ploid-multiploid tumors (p = 0.022). CONCLUSION: Our results suggest that DNA ploidy, as determined by image analysis, provides an independent prognostic parameter for patients with NSCLC and thus, could be used to identify a subset of patients with more aggressive tumors.