The year 2012 sees two papers from IMCB garner more than 1,000 citations each. The number of citations is one indicator of a paper's scientific impact, and in the biological sciences a paper is termed a "Citation Classic" once it has accumulated more than 400 citations. According to ISI# there are 14 such papers published by IMCB groups. Papers with 1,000 citations are exceptional, and in the context of Singapore's relatively new push in biology, only three non-clinical papers have achieved this notable mark (ref 1, 2, 3). Here we highlight IMCB's most highly cited paper from Alan Porter and his lab.
Reiner U. Jänicke1, Michael L. Sprengart2, Mas R. Wati3, Alan G. Porter4.
1 - University of Düsseldorf, Institute of Molecular Medicine, Düsseldorf, Germany.
2 - University of Heidelberg, Germany.
3 - Universiti Brunei Darussalam.
4 - 7 Alexander Avenue, Droitwich, UKWR9 8NH.
Published in The Journal of Biological Chemistry, April 17, 1998 (cited 1,030 times)
When we initiated this project, we knew the zymogen form of the protease caspase-3 (pro-caspase-3) is present in virtually all cells examined. Moreover, caspase-3 is typically proteolytically activated by apoptotic stimuli, leading in turn to its limited and highly specific cleavage of many protein substrates, which in many cases appeared to (or was predicted to) alter their function. It was also known caspase-3 knockout mice have abnormal brain development. But the requirement for caspase-3 in cell death and the classic morphological changes of apoptosis (cell shrinkage, membrane blebbing and DNA fragmentation) was unknown.
Reiner Jänicke first noticed that MCF-7 cells, unlike other tumour cell lines, synthesize no pro-caspase-3 protein. Initially, we thought that this might be due to transcriptional or translational silencing, but Michael Sprengart performed RT-PCR on the CASP-3 mRNA, revealing a substantially truncated mRNA that was shortened by 125 bp only in MCF-7 cells. Spurred on by this intriguing finding, Michael went on to show its genomic DNA also had a deletion. It was clear from these studies that MCF-7 cells had lost caspase-3 protein owing to a 47-bp deletion within exon 3 of the CASP-3 gene. This deletion resulted in the skipping of exon 3 during pre-mRNA splicing, thereby abrogating translation of the CASP-3 mRNA. Importantly, this was not a clonal artefact, but a genuine situation, as the same results were achieved with MCF-7 clones obtained from several other sources including the ATCC.
Reiner then showed that although MCF-7 cells were still sensitive to various death stimuli, no DNA fragmentation occurred in these cells, in contrast to other sensitive cell types that synthesize caspase-3. Equally as interesting, he went on to demonstrate that MCF-7 cells undergoing cell death did not display some of the distinct morphological features typical of apoptotic cells such as shrinkage and membrane blebbing. As all these hallmarks appeared in dying caspase-3-reconstituted MCF-7 cells, we were able to conclude from this study that although caspase-3 is dispensable for apoptosis induced by some death signals, it is required for DNA fragmentation and typical morphological changes of apoptosis including cell shrinkage and membrane blebbing – at least in MCF-7 cells. Although these are surely important features essential for an efficient clearance of apoptotic cells by phagocytes, many questions remained, including: did the loss of caspase-3 occur during tumorigenesis in vivo; and if so, did its loss contribute to the original breast malignancy. At the time, we aimed for a rapid publication rather than publication in a high impact journal. Luckily, this was achieved with J. Biol. Chem., which accepted and published our manuscript within a record time of just over two months without any revisions. Subsequently, our discovery resulted in numerous collaborations worldwide.
In conclusion, at the start of this project, we never dreamed that our paper would be cited over 1,000 times and become the most highly cited IMCB paper in 25 years. Our success is testament to some divergent thinking as well as an inspired team effort in an up to date, important and highly competitive field.
Figure Legend: Caspase-3 is absent from MCF-7 cells and is not essential for TNF- or staurosporine-induced apoptosis.
A, TNF cytotoxicity assays of cells treated for 4 (HeLa D98 and H21) or 20 h (MCF-7) with TNF/Chx or for 16 h with staurosporine
(staurosp.) show caspase-3 is not essential for TNF- or staurosporine-induced apoptosis. B, lack of caspase-3 protein in MCF-7 cells. Shown are Western blot analyses of caspase-3 expression in lysates of untreated cells (lanes 1, 4, and 7), cells treated for 4 (lanes 2 and 5) or 20 h (lane 8) with TNF/Chx, and cells treated for 16 h with staurosporine (lanes 3, 6, and 9).