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CORRESPONDENCE Rathbun et al1 have reported apoptosis in the
Fanconi anemia (FA) cell line HSC536N in response to interferon- We incubated the FANCC mutant cell line HSC536N for 6 hours with different doses of IFN- Cell extracts were subjected to Western blotting and probed with an
antibody for caspase 3 (Pharmingen, catalog no. 65906E, San Diego,
CA) (Figure 1). In contrast to
Rathbun et al, we found substantial cleavage of caspase 3, with
generation of p20 and p17 cleaved forms, with only 10 µg protein
loading. To further investigate the discrepancy with the previous
report, we repeated the blots using the same antibody employed by
Rathbun et al (Transduction Laboratories, C31720, San Diego, CA).
Cleaved forms of caspase 3 were scarcely detectable at 10 µg
loading (Figure 1B). We note that the manufacturer only claims
detection of the proform of caspase 3 by this antibody. Western blots
were also performed with an anti-PARP antibody. In this case we found
no detectable evidence of PARP cleavage, compared to standard controls
(Figure 1). DNA was also extracted for gel electrophoresis, and DNA
laddering was not detected.
Are these cells really undergoing apoptosis? The primary definition of apoptosis is based on morphology. Examination of Giemsa-stained cytocentrifuge preparations of anti-Fas-treated cells clearly showed classical apoptosis, with nuclear condensation, and chromatin marginalization and fragmentation. We conclude that HSC536 cells do indeed undergo morphological apoptosis in response to Fas antibodies and that the failure of Rathbun et al to detect quantitative caspase 3 cleavage was a technical problem associated with the antibody. On the other hand, even if the minor band seen in their blots is a PARP cleavage product, cleavage of PARP within these cells is still negligible. There is clear evidence that primary FA cells are hypersensitive to
IFN-
Tim R. Rutherford, Nyree E. Myatt, Frances M. Gibson, and Alan A. Clarke Supported by a grant from the United Kingdom Leukaemia Research Fund References
1.
Rathbun RK, Christianson TA, Faulkner GR, et al.
Interferon-gamma-induced apoptotic responses of Fanconi anemia group C hematopoietic progenitor cells involve caspase 8-dependent activation of caspase 3 family members.
Blood.
2000;96:4204-4211 2. Clarke AA, Philpott NJ, Gordon-Smith EC, Rutherford TR. The sensitivity of Fanconi anaemia group C cells to apoptosis induced by mitomycin C is due to oxygen radical generation, not DNA crosslinking. Br J Haematol. 1997;96:240-247[CrossRef][Medline] [Order article via Infotrieve]. 3. Guillouf C, Wang TS, Liu J, et al. Fanconi anemia C protein acts at a switch between apoptosis and necrosis in mitomycin C-induced cell death. Exp Cell Res. 1999;246:384-394[CrossRef][Medline] [Order article via Infotrieve].
4.
Philpott NJ, Turner AJ, Scopes J, et al.
The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques.
Blood.
1996;87:2244-2251
5.
Pang Q, Fagerlie S, Christianson TA, et al.
The Fanconi anemia protein FANCC binds to and facilitates the activation of STAT1 by gamma interferon and hematopoietic growth factors.
Mol Cell Biol.
2000;20:4724-4735
Response:Apoptosis and FANCCRutherford et al have stated that in our paper on caspase activation in Fanconi anemia (FA)1 cells "the classic assays of apoptosis" were not used. They are incorrect. Figure 3 was derived from experiments using the TUNEL assay. An article by our group that was published in Blood a year earlier2 also reported our experience using both morphology and TUNEL assays. In both papers we demonstrated that FA cells are excessively apoptotic. Since the publication of the paper in question we have published additional papers that have further clarified the molecular nature of the apoptotic FA-C phenotype3-8 and have identified a pivotal control point for caspase 3 activation in FA cells.6,7 IFN itself cannot fully reveal a differentially hypersensitive FA
phenotype in the isogenic cell lines. We reported this a number of
years ago when we noted that a blocking antibody to fas abrogated
apoptotic responses of FA-C cells to IFN In our hands, the most reliable and quantitative assessments of the
induced apoptotic phenotype in FA cells (murine or human cells) are
made using a flow cytometric method that permits quantification of
single cells containing caspase 3.6-8 Using this method,
TNF Rutherford et al have stated that "in order to show cleavage," our group "had to load 100 µg of protein." This, too, is incorrect. At no time have we stated that detection of cleavage of either PARP or caspase 3 required a 100 µg load. Also, the authors' statements that "in contrast to Rathbun et al, we found substantial cleavage of caspase 3...with only 10 µg loading," and "failure of Rathbun et al to detect quantitative caspase 3 cleavage..." are misleading. Their observation does not stand in contrast to ours. We consistently find differential caspase 3 activation and over the past several years have shown that caspase 3 activation can be shown at a single-cell level, a quantitative standard that simply cannot be matched by Western blotting. Indeed, the result reported here by Rutherford et al at once confirms our results and clarifies that the anti-PARP antibody in question is capable of detecting the cleaved form of the molecule. While we have not seen differential PARP activation in isogenic cell lines, we view the immunoblot method as insensitive and have not focused much attention on PARP recently because we now recognize that PARP cleavage is not necessary for execution of an apoptotic program.9 The authors' claim that FA cell lines treated with MMC do not undergo "normal apoptosis" is difficult to understand in part because the only cited reference that used isogenic FA-C cells did, in fact, demonstrate clearly that PARP cleavage induced by MMC was noted only in mutant cells, not in FANCC corrected cells.10(Fig5C) Moreover, work from our laboratory published in this journal shows a major reduction in TUNEL-positive MMC-exposed cells in FANCC-complemented FA-C cells.2(Fig6A) Of equal relevance we have also noted (G.C.B. and R. K. Rathbun, unpublished observations, September 2000) that MMC-exposed FA cells have more cleaved caspase 9 than do MMC-exposed FA-C cells complemented with FANCC. Because there is not much disagreement on the involvement of caspase 3 in the FANCC pathway, it is probable that all of us share the view that control points for caspase 3 activation are critically important for an understanding of FA cells. We have chosen this path.1,3,5-8 What are we left with? We are possibly left with some evidence that internucleosomal cleavage of chromatin is somehow different in FA cells treated with MMC, but we are not left with any viable notion that MMC does not induce a fully loaded apoptotic response. In summary, Rutherford et al have attributed to our report specific deficiencies that did not exist. If their initial intention was to reproduce our work, their group should have done the same experiments. In fact, they failed to reproduce conditions we used (TUNEL positivity is most substantial after 48 hours of exposure to IFN and agonistic fas antibody2[Fig3]), failed to use isogenic sets of FA mutant cells, and used a method for quantifying apoptotic cells that is not fully validated for studies on Fanconi cells, when compared to results from a battery of conventional measures of apoptosis, methods they themselves claim to be gold standards. Their concluding suggestion, "high constitutive levels of fas expression on FA cells" may account for some effects we have all seen, has been tested by us years ago and ruled out at least in B-cell lines.4 When the authors test their next hypothesis, they should utilize isogenic FA cell lines now widely extant, use gold standard apoptosis assays, use the combinations of cytokines now known to induce maximal differential apoptosis in FA cells, and consider using caspase 3-activation assays that can detect biologically meaningful fractional changes in cell populations. If they do these things, we have no doubt they will find that FA-C cells exhibit exaggerated responses that are both statistically and biologically significant. We suppose that one might spend a good bit of time arguing about certain semantic issues that have evolved in this field, issues that might be likened to the tree-falling-in-the-forest-unseen argument (eg, if all other molecular events characteristic of apoptosis occur, does a reduced detectability of internucleosomally cleaved DNA mean that there is no "apoptosis?"). Moving beyond such arguments, we are confident that most workers in this field, including the 2 parties disagreeing here, will ultimately concur that FA-C cells are more poised to undergo programmed cell death when challenged with certain chemical agents or selected extracellular biologic cues and that the apoptotic response of these cells is generally exaggerated because the protein encoded by the FANCC gene modulates such responses.
Grover C. Bagby, Jr
References 1. Rathbun RK, Christianson TA, Faulkner GR, et al. Interferon-g-induced apoptotic responses of Fanconi anemia group C hematopoietic progenitor cells involve caspase 8-dependent activation of caspase 3 family members. Blood. 2000;96:4204-4211.
2.
Rathbun RK, Faulkner GR, Ostroski MH, et al.
Inactivation of the Fanconi anemia group C (FAC) gene augments interferon-gamma-induced apoptotic responses in hematopoietic cells.
Blood.
1997;90:974-985
3.
Fagerlie SR, Diaz J, Christianson TA, et al.
Functional correction of FA-C cells with FANCC suppresses the expression of interferon gamma-inducible genes.
Blood.
2001;97:3017-3024 4. Koh PS, Hughes GC, Faulkner GR, Keeble WW, Bagby GC. The Fanconi anemia group C gene product modulates apoptotic responses to tumor necrosis factor-a and Fas ligand but does not suppress expression of receptors of the tumor necrosis factor receptor superfamily. Exp Hematol. 1999;27:1-8[CrossRef][Medline] [Order article via Infotrieve]. 5. Pang Q, Fagerlie S, Christianson TA, et al. The Fanconi anemia protein FANCC binds to and facilitates the activation of STAT1 by gamma interferon and hematopoietic growth factors. Mol Cell Biol. 2000;20:4724-4735.
6.
Pang Q, Keeble W, Diaz J, et al.
Role of double-stranded RNA-dependent protein kinase in mediating hypersensitivity of Fanconi anemia complementation group C cells to interferon gamma, tumor necrosis factor-alpha, and double-stranded RNA.
Blood.
2001;97:1644-1652 7. Pang Q, Keeble W, Christianson TA, Faulkner GR, Bagby GC. FANCC interacts with hsp70 to protect hematopoietic cells from IFNg/TNFa-mediated cytotoxicity. EMBO J. 2001;20:4478-4489[CrossRef][Medline] [Order article via Infotrieve].
8.
Pang Q, Christianson TA, Keeble W, et al.
The Fanconi anemia complementation group C gene product: structural evidence of multifunctionality.
Blood.
2001;98:1392-1401
9.
Wang Z-Q, Stingl L, Morrison C, et al.
PARP is important for genomic stability but dispensable in apoptosis.
Genes Dev.
1997;11:2347-2358 10. Guillouf C, Wang TS, Liu J, et al. Fanconi anemia C protein acts at a switch between apoptosis and necrosis in mitomycin C-induced cell death. Exp Cell Res. 1999;246:384-394. 11. Clarke AA, Philpott NJ, Gordon-Smith EC, Rutherford TR. The sensitivity of Fanconi anaemia group C cells to apoptosis induced by mitomycin C is due to oxygen radical generation, not DNA crosslinking. Br J Haematol. 1997;96:240-247   CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
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and does not cleave PARP in response to Fas-mediated
cell killing
(IFN-
, or IFN
