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Immunofluorescence studies on the cell cycle expression of cyclin A and cyclin E

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posted on 2024-09-02, 23:41 authored by Fredrik Erlandsson

Cell cycle progression is tightly regulated in normal eucaryotic cells. Cancer is caused by the transformation of cells, and is always associated with defects in the cell cycle regulatory machinery. An important result of a defective cell cycle regulation is genetic instability, as the cell with a deficient cell cycle control fails to correctly duplicate and split its genome. Genetic instability leads to the acquisition of new characteristics, and is thought to cause tumour progression. Cyclin Dependent Kinases (CDKs), and their regulatory subunits, the cyclins, are central in cell cycle regulation. Cell cycle progression is accomplished by the successive activation and deactivation of the various cyclin-CDK complexes. CyclinCDK complexes act by phosphorylating a range of target proteins appropriate for the cell cycle phase in which the cyclin-CDK complex is active. Cyclin E-CDK2 is active in late G1 and phosphorylates proteins central to the progression through late G1 and entry into S-phase. Cyclin A-CDK2 is active in S and G2, and cyclin A-CDK2 activity is essential from progression through S-phase, and entry into mitosis.

The study of the cell cycle machinery puts high demand on the methodological approach, as cell cycle progression often is altered, or even abrogated, by the treatment of the cells required by many of the traditional biochemical methods. In order to study the intricate cell cycle regulatory mechanisms a novel method based on immunofluorescence staining was developed which allowed the semi-quantitative measurement of the levels of a large number of proteins in individual cells in culture or tissue samples.

Cyclin A and cyclin E are both central in the regulation of DNA replication. Hence a deregulated expression of either cyclin could potentially cause genetic instability. In order to study the expression patterns of cyclin A and cyclin E over the cell cycle in normal and transformed cells a triple immunofluorescence staining protocol was utilised. The position in the cell cycle of each individual cell could then be established, and correlated with the immunofluorescence staining intensity for cyclin A or cyclin E.

Nuclear cyclin A accumulation was shown to begin virtually exactly as the cells entered Sphase, i e very close to the G1/S-transition. The cyclin A accumulation began at the same point in the cell cycle and progressed with similar kinetics in both normal and transformed cells. The data suggests that deregulation of cyclin A expression is not commonly occurring in transformed cells, possibly because it is deleterious.

Cyclin E accumulation, on the other hand, was shown to be highly different in normal and transformed cells. In normal cells cyclin E levels were found to rise after progression through the R-point, peak in late G1-phase, and then decrease as the cells entered S-phase. In transformed cells cyclin E accumulation commonly continued throughout S-phase, and cyclin E was often not completely degraded until in mitosis. Therefore the clinical implications of a deregulated cyclin E expression pattern in cervical carcinoma lesions was investigated. It was found that a highly deranged cyclin E expression over the cell cycle was associated with poor survival. The data are well in line with the results presented by others, which have shown that a deregulated cyclin E expression can cause genetic instability.

List of scientific papers

I. Wahlby C, Erlandsson F, Bengtsson E, Zetterberg A (2002). Sequential immunofluorescence staining and image analysis for detection of large numbers of antigens in individual cell nuclei. Cytometry. 47(1): 32-41.
https://pubmed.ncbi.nlm.nih.gov/11774347

II. Erlandsson F, Linnman C, Ekholm S, Bengtsson E, Zetterberg A (2000). A detailed analysis of cyclin A accumulation at the G(1)/S border in normal and transformed cells. Exp Cell Res. 259(1): 86-95.
https://pubmed.ncbi.nlm.nih.gov/10942581

III. Erlandsson F, Wahlby C, Ekholm-Reed S, Hellstrom AC, Bengtsson E, Zetterberg A (2003). Abnormal expression pattern of cyclin E in tumour cells. Int J Cancer. 104(3): 369-75.
https://pubmed.ncbi.nlm.nih.gov/12569561

IV. Erlandsson F, Martinsson HS, Wallin KL, Andersson S, Hellström AC, Zetterberg A (2003). Cell cycle expression of cyclin E in neoplastic lesions of the uterine cervix. [Manuscript]

History

Defence date

2004-04-28

Department

  • Department of Oncology-Pathology

Publication year

2004

Thesis type

  • Doctoral thesis

ISBN-10

91-7349-809-2

Number of supporting papers

4

Language

  • eng

Original publication date

2004-04-07

Author name in thesis

Erlandsson, Fredrik

Original department name

Department of Oncology-Pathology

Place of publication

Stockholm

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