An all solidstate nearinfrared timecorrelated single photon counting instrument for dynamic lifetime measurements in DNA sequencing applications Rev.Single quantum well light emitting diodes demonstrated as excitation sources for nanosecond phasemodulation fluorescence lifetime measurements Rev.
Bcl-2 protein expression has been found to block apoptosis and its overexpression has been implicated in lymphoid malignancies where the chromosomal translocation t(14;18) is present. In this study we investigated bcl-2 transcription and protein expression in cultured cervical carcinoma cell lines and keratinocytes. Western blotting and immunofluorescence microscopy demonstrated bcl-2 expression in the cytoplasm of 4 out of 5 cervical carcinoma cell lines examined (HeLa, CaSki, C-33A, and HT-3, but not SiHa). Bcl-2 protein expression was undetectable in normal keratinocytes. None of the cell lines examined demonstrated chromosomal translocation or rearrangement at the major breakpoint-cluster region (MBR) of the bcl-2 gene using either Southern blot or polymerase chain reaction (PCR) analyses. Northern blot analysis demonstrated low levels of bcl-2 transcription in HeLa, CaSki, and C-33A cell lines while reverse transcriptase (RT)-PCR demonstrated bcl-2 transcription in all cervical carcinoma cell lines which had bcl-2 protein expression. Thus, these data suggest that bcl-2 expression occurs in cervical carcinoma cell lines in the absence of chromosomal translocation or rearrangement of the bcl-2 gene. However, each of these cervical carcinoma cell lines contains inactive p53, either due to mutation (C-33A and HT-3) or via complexation and degradation with human papillomavirus (HPV) 16/18 E6 protein (HeLa and CaSki). Thus, functional p53, which can induce apoptosis in certain cells, is not present in these cervical cells which have increased bcl-2 expression. Increased bcl-2 expression under conditions of p53 inactivation may provide cells with a selective advantage for survival and consequently play a role in the development of cervical carcinogenesis.
Recent technological advances have provided an opportunity for the development of "Fluorescence Lifetime Imaging Microscopy" (FLIM). FLIM is an extremely important advance, as it allows for the first time, the sensitivity of the fluorescence lifetime to environmental parameters to be monitored in a spatial manner in single living cells. FLIM can be developed both on conventional and confocal fluorescence microscopes. Fluorescence lifetime detection can be performed using either time-or frequency-domain methods. In this paper, we report on the development of conventional and confocal FLIM systems currently underway in our laboratory. We also present three examples of current biological research projects in which we employ FLIM.
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