Characterization of keratin and cell cycle protein expression in cell lines from squamous intraepithelial lesions progressing towards a malignant phenotype

Objective: This study was designed to analyze the combinatorial chemotherapeutic effect of temozolomide (TMZ), the most common drug in glioblastoma treatment and a purified carbohydrate (Fr-II) from the edible mushroom Pleurotus florida, on human glioblastoma cell lines.Methods: Fr-II was purified by size-exclusion chromatography and characterised by different mass spectroscopy analysis. Human glioblastoma cells were treated with TMZ, Fr-II, and combination of TMZ and Fr-II. Cell cytotoxicity was measured by MTT assay, cell cycle phase distribution was determined by cell cycle analysis and followed by the relative p53 protein expression was analyzed by western blot analysis.Results: Chemical analysis of Fr-II confirmed the glycosidically linked two units of glucose with terminally attached mannitol with mass of 506 Da. Fr-II treatment exhibited cytotoxicity in both the cell lines in a dose-dependent manner with most effective dose at 200µg/ml. When Fr-II (200µg/ml) was combined with a dose range of TMZ it showed a more cellular cytotoxicity compared to the cytotoxicity of TMZ alone with most oppressive combinatorial dose at 400µM (TMZ)+200µg/ml (Fr-II). In compliance, with the above results, both cell lines showed a 10% increase in no. of cells (p<0.05) in G2/M phase indicating an arrest of cell cycle and increased p53 protein expression (p<0.05) at the combinatorial dose than TMZ alone at 400µM, but Fr-II alone didn’t show any cell cycle arrest nor did it show increased p53 expression.Conclusion: Therefore it confirms that Fr-II synergizes with TMZ to significantly intensify its anti-proliferative properties, thereby emerging as an effective element for combinatorial treatment of glioblastoma.

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S100 protein expression in human melanoma cells: Comparison of levels of expression among different cell lines and individual cells in different phases of the cell cycle

Experimental Cell Research ◽

10.1016/0014-4827(90)90116-r ◽

1990 ◽

Vol 187 (1) ◽

pp. 59-64 ◽

Cited By ~ 28

Author(s):

A. Marks ◽

D. Petsche ◽

D. O'Hanlon ◽

P.C. Kwong ◽

R. Stead ◽

...

Keyword(s):

Cell Cycle ◽

Protein Expression ◽

Cell Lines ◽

S100 Protein ◽

Melanoma Cells ◽

Human Melanoma ◽

Human Melanoma Cells

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O85 Cell cycle protein expression patterns in HPV-containing and -lacking tonsillar squamous cell carcinomas and association with prognosis: a role for p21?

Oral Oncology Supplement ◽

10.1016/s1744-7895(07)70129-3 ◽

2007 ◽

Vol 2 (1) ◽

pp. 84

Author(s):

J. Mooren ◽

J. Manni ◽

H. Hafkamp ◽

B. Klingenberg ◽

A. Voogd ◽

...

Keyword(s):

Cell Cycle ◽

Protein Expression ◽

Squamous Cell ◽

Expression Patterns ◽

Squamous Cell Carcinomas ◽

Cell Cycle Protein

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Evaluation of proliferative index and cell cycle protein expression in choroid plexus tumors in children

Acta Neuropathologica ◽

10.1007/s004010100419 ◽

2001 ◽

Vol 103 (1) ◽

pp. 1-10 ◽

Cited By ~ 42

Author(s):

Carlos G. Carlotti ◽

Bodour Salhia ◽

Sheila Weitzman ◽

Mark Greenberg ◽

Peter B. Dirks ◽

...

Keyword(s):

Cell Cycle ◽

Protein Expression ◽

Choroid Plexus ◽

Proliferative Index ◽

Cell Cycle Protein ◽

Choroid Plexus Tumors

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Protein Expression Patterns of Candidate Genes Involved in Apoptosis and Cell Cycle Control in Genetic Subgroups of Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v104.11.2796.2796 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2796-2796

Author(s):

Christof Schneider ◽

Dirk Winkler ◽

Meike Loddenkemper ◽

Alexander Krober ◽

Peter Lichter ◽

...

Keyword(s):

Cell Cycle ◽

Protein Expression ◽

Cyclin D1 ◽

Candidate Genes ◽

Cell Lines ◽

Normal Karyotype ◽

Lymphocytic Leukemia ◽

Expression Levels ◽

Protein Levels ◽

Atm Protein

Abstract Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a highly variable clinical course. Genomic aberrations (such as 13q−, 11q−, +12q, 17p−) can be found in about 80% of CLL cases and define pathogenic as well as clinical subgroups. Similarly, the mutational status of the variable region of the immunoglobulin heavy-chain gene (VH) identifies subgroups with different maturation stage and clinical outcome. In this study protein expression levels of candidate genes involved in cell cycle and apoptosis control (p53, ATM, Akt1, PI3-K, p21, p27, cdk4, Cyclin-D1, D2, D3, Bax, Bcl-2, Apaf-1, Smac, XIAP, cIAP2, survivin) were examined by Western Blotting. A total of 87 CLL cases derived from the subgroups with 11q- (n=22), 17p-/p53 mutation (n=18), +12q (n=24), 13q- (n=8) or a normal karyotype (n=15) were studied and compared to the cell lines EHEB and JVM-2. VH-mutation status was available for 65 cases (unmutated n=48, mutated n=17). Due to limitations in sample availability not all proteins could be examined in all cases. A highly homogenous expression pattern for all the proteins studied was observed in the CLL subgroup with a normal karyotype. This pattern was independent of the VH-status. CLL samples with normal karyotype, +12q and 13q deletion showed equal levels of ATM as compared to EHEB and JVM-2. As compared to cases with a normal karyotype the ATM level within the 11q- subgroup was reduced in 5 cases and absent in 1 case among 11 evaluable 11q- cases. The 17p- subgroup was comprised of 3 cases with concomitant 17p- and 11q- and 15 cases with 17p- but no 11q-. The latter group showed ATM protein levels comparable to the levels of the normal karyotype group. In the group with 17p- and 11q- there was an ATM expression level similar to the groups with 17p- and normal karyotype in two cases while one case had a reduced ATM protein level comparable to the 11q- subgroup. All cases with 17p- exhibited a stronger expression of p53 as compared to the cell lines and all other cases, except for one case with normal karyotype and one with an 11q-. No p53 mutations could be detected in exons 5–9 by sequencing in these two cases. High levels of survivin protein were found in all cases with 17p- and/or 11q-, 13q-, +12q while the subgroup with a normal karyotype showed lower levels. High levels of cdk4 protein were expressed in cases with 17p-, 11q- and 13q- while cdk4 protein levels were low in the subgroup with +12q and normal karyotype. Regarding p21, p27, Bcl2, Bax, Smac, Apaf-1, Cyclin D1–D3, cIAP2, XIAP, Akt1 and PI3K no variation in the expression levels were observed across the genetic CLL subgroups. Comparing the CLL cases to the cell lines the differences in expression levels were found for the cell cycle regulators Cyclin D1, D2, D3, p21 and p27. While the cell lines showed strong protein levels for Cyclin D1, D2, D3 and p21, they were nearly absent in the CLL cases. Expression of p27 was higher in all CLL cases as compared to JVM-2 and EHEB. In conclusion, the 17q- subgroup was the only group with a high level of p53 protein expression indicating that p53 is the affected gene in this subgroup. In contrast, the ATM protein levels are reduced only in a part of the 11q- cases indicating a possible role of additional candidate genes. Cases with +12q and normal karyotype showed weak expression of cdk4 pointing out a possible function in these subgroups.

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Truncation in CCND1 mRNA 3′ UTR Is Associated with An Aggressive Phenotype and Chemoresistance

Blood ◽

10.1182/blood.v112.11.5287.5287 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5287-5287

Author(s):

Robert W Chen ◽

Myo Htut ◽

Britta Hoehn ◽

Eamon Berge ◽

William Robinson ◽

...

Keyword(s):

Cell Cycle ◽

Protein Expression ◽

Cyclin D1 ◽

Cell Lines ◽

Cell Cycle Progression ◽

Serum Starvation ◽

Cycle Progression ◽

Serum Free ◽

Aggressive Phenotype ◽

Free Media

Abstract Mantle Cell Lymphoma (MCL) represents 5–10% of all non-Hodgkins lymphomas, making it an uncommon but difficult form of lymphoma to treat. It has the worst prognosis among the B cell lymphomas with median survival of three years. The genetic hallmark of MCL is the t(11,14)(q13;32) translocation causing amplification of cyclin D1 (CCND1). It is a well known cell cycle regulator. Multiple reports have shown a truncation in the cyclin D1 mRNA 3′ untranslated region. This truncation increases CCND1 protein expression by not only enhancing the half-life of CCND1 mRNA, but also evades microRNA regulation of mRNA translation. The dramatic overexpression of CCND1 mRNA and protein has been associated to poor clinical outcome in patients. We hypothesize that this truncation leads to a more aggressive phenotype and induces chemoresistance in MCL. We have identified 4 MCL cell lines (Granta-519, JVM-2, Jeko-1, and Z138) with different levels of the truncated CCND1 mRNA. We were able to show that Z138 and Jeko-1 have a much higher ratio of truncated CCND1 mRNA to the full length CCND1 mRNA as compared to Granta-519 and JVM-2. We were also able to show that this truncated mRNA leads to an increase in CCND1 protein expression. By using flow cytometry, we correlated the increase in CCND1 protein expression to faster cell cycle progression. We proposed that cell lines with increased CCND1 expression are phenotypically more aggressive, and would be able to continue cell cycle progression without serum support. We were able to arrest JVM-2 in G1 phase after 48 hours of serum starvation. However, we were not able to arrest cell cycle progression in Jeko-1 even after 96 hours of serum starvation. Western blot analysis shows that CCND1 protein expression is decreased in JVM-2 but remains unchanged in Jeko-1 with serum starvation. The same phenomenon was observed in Granta-519 and Z138. The MCL cell lines (Jeko-1 and Z-138) with more CCND1 protein expression were able to continue cell cycle progression in serum free media. The MCL cell lines (JVM-2 and Granta-519) with less CCND1 protein expression were not able to continue cell cycle progression in serum free media. This shows that CCND1 overexpression is associated with a more aggressive phenotype. We then treated the 4 MCL cell lines with varying concentrations of doxorubicin, a standard anthracycline chemotherapy used in the treatment of MCL patients. We used MTS assay to assess cell proliferation after treatment with doxorubicin. We found the IC 50 (inhibitory concentration 50%) of doxorubicin in these cell lines varied from 6nM to 600nM. The cell lines (Jeko-1 and Z-138) with more CCND1 protein expression have a much higher IC 50 as compared to the cell lines (JVM-2 and Granta-519) with less CCND1 protein expression. This demonstrates that CCND1 overexpression is associated with chemoresistance. We conclude truncation in CCND1 mRNA leads to increased CCND1 protein expression and faster cell cycle progression CCND1 overexpression is associated with an aggressive phenotype CCND1 overexpression is associated with chemoresistance.

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Effect of the specific P53 stabilizer CBS9106 on multiple myeloma (MM)

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.8601 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. 8601-8601

Author(s):

H. Ikeda ◽

T. Hideshima ◽

G. Perrone ◽

Y. Okawa ◽

N. Raje ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Protein Expression ◽

Tumor Cells ◽

Cell Lines ◽

Primary Tumor ◽

P53 Protein ◽

Wild Type ◽

Cycle Arrest ◽

P53 Protein Expression

8601 Background: The mutations of P53 tumor suppressor protein are associated with progressive in Multiple Myeloma (MM), conversely, stabilization of P53 leads to cell cycle arrest and apoptosis. In this study, we examined p53 protein expression and demonstrated the effect of P53 stabilization using a novel specific P53 stabilizer CBS9106 in MM. Method: We examined P53 protein expression using Immunoblot analysis, as well as the growth inhibitory effect of CBS9106 in MM cell lines and primary tumor cells from MM patients. We also defined whether CBS9106 can overcome the growth promoting effect of exogenous cytokines and bone marrow stroma cells (BMSCs) using [3H]-thymidine uptake assay. Results: Expression of P53 protein was observed in 3/3 primary tumor cells from MM patients and 6/6 MM cell lines. CBS9106 at low nM levels triggered cytotoxicity against p53 wild type MM cell lines and primary tumor cells from MM patients, associated with phosphorylation of P53 (serine15 and 20). In contrast, CBS9106 did not affect the survival of normal peripheral blood mononuclear cells from healthy volunteers at concentrations as high as 10 μM. This agent also induced G1 cell cycle arrest, followed by apoptosis associated with cleavage of caspase-3, -8, -9 and PARP. Neither growth stimulating cytokines (IL-6 and IGF-1) nor BMSCs protected against apoptotic effect of CBS9106. Moreover, we demonstrate that combination of CBS9106 with MDM2 inhibitor Nutrin3 or proteasome inhibitor bortezomib induces synergistic anti-MM activity in both P53 wild type MM cell lines and primary tumor cells from MM patients. Conclusions: Stabilizing P53 by CBS9106 represents a novel promising p53-based therapy in MM. These results provide the preclinical framework supporting evaluation of CBS9106 in clinical trials to improve patient outcome in MM. No significant financial relationships to disclose.

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