J.P. Doherty

Many strains of E. coli K12 restrict DNA containing cytosine methylation such as that present in plant and animal genomes. Such restriction can severely inhibit the efficiency of cloning genomic DNAs. We have quantitatively evaluated a total of 39 E. coli strains for their tolerance to cytosine methylation in phage and plasmid cloning systems. Quantitative estimations of relative tolerance to methylation for these strains are presented, together with the evaluation of the most promising strains in practical recombinant cloning situations. Host strains are recommended for different recombinant cloning requirements. These data also provide a rational basis for future construction of 'ideal' hosts combining optimal methylation tolerance with additional advantageous mutations.

We have investigated the function and sequence specificity of DNA methylation in the hypermethylated CpG island promoter region of the endogenous human LINE-1 (L1) retrotransposon family. In nontransformed human embryonic fibroblasts, inhibition of DNA methylation with 5-azadeoxycytidine induced a greater than 4-fold increase in transcription from potentially functional L1 elements without increasing the transcription level of the majority of degenerate elements, implicating hypermethylation in the repression of L1 activity. Using bisulfite genomic sequencing to assess the pattern of methylation in a subset of nondegenerate L1 elements, we found 29 sites within a 460-base pair region of the noncoding (top) DNA strand of the L1 promoter in which cytosine methylation was maintained with high efficiency. Of these, 25 were at CG dinucleotides and four were in non-CG sites. When the methylation sites were analyzed for the complementary (bottom) strand, the only highly conserved sites of methylation were in CG dinucleotides. Several of these sites of CG methylation in the bottom (coding) strand were at positions where top (noncoding) strand-derived sequences were unmethylated, suggesting that these sites might be maintained in a hemi-methylated state. Hence, there is a subset of human L1 elements in which methylation is efficiently maintained in asymmetric non-CG sites and further that this non-CG methylation may be part of a wider phenomenon involving hemi-methylation at CG dinucleotides. Maintenance of asymmetric methylation at non-CG sites (and possibly at hemi-methylated CG dinucleotides) could be through a novel DNA methyltransferase activity. Alternatively, the promoter region of L1 elements may be induced by factor binding to form some type of secondary structure that presents as a highly efficient substrate for de novo methylation.

Hsp72 protects cells against apoptosis in response to various stresses. By simultaneously measuring cytochrome c localization and nuclear morphology in mouse embryo fibroblasts, we have shown that Hsp72 blocks cytochrome c release from mitochondria in response to cytotoxic stress and that permeabilization of the outer mitochondrial membrane is the critical point in deciding the fate of the cell. Hsp72 did not inhibit apoptosis in mouse embryo fibroblasts once cytochrome c had been released from the mitochondria. Recent reports have claimed that Hsp72 can prevent caspase activation by inhibiting the oligomerization of Apaf-1 in the presence of cytochrome c and dATP. We now show that this apparent function of recombinant Hsp72 is due to the presence of salt in the Hsp72 preparation and that the same response can be achieved by the addition of heat-denatured Hsp72 in the same high salt buffer or by the high salt buffer alone. Hsp72 expressed in a range of different cell lines had no inhibitory effect on cytochrome c-stimulated caspase activity of cytosolic extracts. We conclude that the protective effect of Hsp72 occurs upstream of the mitochondria and not through the inhibition of the apoptosome. Hsp72 protects cells against apoptosis in response to various stresses. By simultaneously measuring cytochrome c localization and nuclear morphology in mouse embryo fibroblasts, we have shown that Hsp72 blocks cytochrome c release from mitochondria in response to cytotoxic stress and that permeabilization of the outer mitochondrial membrane is the critical point in deciding the fate of the cell. Hsp72 did not inhibit apoptosis in mouse embryo fibroblasts once cytochrome c had been released from the mitochondria. Recent reports have claimed that Hsp72 can prevent caspase activation by inhibiting the oligomerization of Apaf-1 in the presence of cytochrome c and dATP. We now show that this apparent function of recombinant Hsp72 is due to the presence of salt in the Hsp72 preparation and that the same response can be achieved by the addition of heat-denatured Hsp72 in the same high salt buffer or by the high salt buffer alone. Hsp72 expressed in a range of different cell lines had no inhibitory effect on cytochrome c-stimulated caspase activity of cytosolic extracts. We conclude that the protective effect of Hsp72 occurs upstream of the mitochondria and not through the inhibition of the apoptosome. Hsp72 belongs to an ancient family of proteins that play a role in many different cellular processes, including the folding of newly synthesized proteins, protein trafficking across cellular membranes, and the assembly/disassembly of protein complexes (1Hartl F.U. Martin J. Curr. Opin. Struct. Biol. 1995; 5: 92-102Crossref PubMed Scopus (154) Google Scholar, 2Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3607) Google Scholar). Less well understood is the ability of Hsp72 to protect cells from cell death. Hsp72 has been shown to protect cells from necrosis and is capable of protecting and repairing important cell metabolic processes (3Gabai V.L. Kabakov A.E. FEBS Lett. 1993; 327: 247-250Crossref PubMed Scopus (55) Google Scholar, 4Kabakov A.E. Gabai V.L. Exp. Cell Res. 1995; 217: 15-21Crossref PubMed Scopus (48) Google Scholar, 5Yaglom J.A. Ekhterae D. Gabai V.L. Sherman M.Y. J. Biol. Chem. 2003; 278: 50483-50496Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 6Li L. Shen G. Li G.C. Exp. Cell Res. 1995; 217: 460-468Crossref PubMed Scopus (22) Google Scholar). It has also been shown to inhibit apoptosis, whereby cells actively engineer their own destruction in response to extracellular signals and intracellular damage (7Samali A. Robertson J.D. Peterson E. Manero F. van Zeijl L. Paul C. Cotgreave I.A. Arrigo A.P. Orrenius S. Cell Stress Chaperones. 2001; 6: 49-58Crossref PubMed Scopus (149) Google Scholar, 8Buzzard K.A. Giaccia A.J. Killender M. Anderson R.L. J. Biol. Chem. 1998; 273: 17147-17153Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 9Gabai V.L. Meriin A.B. Mosser D.D. Caron A.W. Rits S. Shifrin V.I. Sherman M.Y. J. Biol. Chem. 1997; 272: 18033-18037Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 10Mosser D.D. Caron A.W. Bourget L. Denis-Larose C. Massie B. Mol. Cell. Biol. 1997; 17: 5317-5327Crossref PubMed Scopus (872) Google Scholar, 11Jaattela M. Wissing D. Kokholm K. Kallunki T. Egeblad M. EMBO J. 1998; 17: 6124-6134Crossref PubMed Scopus (620) Google Scholar). In contrast to necrosis, which is caused by metabolic failure leading to cell swelling and the eventual rupture of the cell membrane, apoptosis requires active cell metabolism to degrade the nuclear DNA, divide the cell into smaller membrane-bound vesicles, and mark these for phagocytosis by surrounding healthy cells (12Adams J.M. Genes Dev. 2003; 17: 2481-2495Crossref PubMed Scopus (680) Google Scholar, 13Lawen A. Bioessays. 2003; 25: 888-896Crossref PubMed Scopus (355) Google Scholar). Apoptosis commonly involves the activation of caspases, a family of intracellular proteases normally present as zymogens (14Wolf B.B. Green D.R. J. Biol. Chem. 1999; 274: 20049-20052Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar). These caspases commit a cell to apoptosis by activating proteins directly involved in the apoptotic process, or inactivating anti-apoptotic proteins and those responsible for normal homeostasis and repair. Apoptosis through extracellular signaling (the extrinsic pathway) and in response to intracellular damage (the intrinsic pathway) are both dependent on caspase activation but differ in how that activation is achieved. In the case of the extrinsic pathway, membrane-bound receptors are oligomerized in response to ligand binding and directly activate the initiator caspases, caspase-8 and -10 (15Muzio M. Stockwell B.R. Stennicke H.R. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1998; 273: 2926-2930Abstract Full Text Full Text PDF PubMed Scopus (885) Google Scholar). These initiator caspases are then responsible for activating the effector caspases 3, 6, and 7 (16Sun X.M. MacFarlane M. Zhuang J. Wolf B.B. Green D.R. Cohen G.M. J. Biol. Chem. 1999; 274: 5053-5060Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar). In some cells (known as type I cells) this is sufficient to induce apoptosis. In type II cells death is dependent on the release of cytochrome c and other pro-apoptotic factors from the mitochondria in response to caspase-8 activity (17Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K.M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2633) Google Scholar). This mitochondrial pathway is the same pathway activated in the cell in response to intracellular damage. Cytochrome c is released from the intermembrane space of the mitochondria and is bound by Apaf-1 along to a protein as the K. C. G. X.M. Cohen G.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Martin Green D.R. D.D. EMBO J. 1997; PubMed Scopus Google Scholar, D. M. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). This then and which is responsible for activating the effector caspases in a to that achieved by caspase-8 D. M. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). of proteins are to inhibit apoptosis in by the activation of the of these are the anti-apoptotic of that by inhibiting the release of cytochrome c from the mitochondria J.M. S. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). family of apoptotic is the that directly inhibit caspase activity by binding to the active of the Salvesen G.S. EMBO J. 1997; PubMed Scopus Google Scholar, Salvesen G.S. Nature. 1997; PubMed Scopus Google Scholar). Hsp72 also apoptosis but the by which this is achieved have that Hsp72 the of an active by binding directly to Apaf-1 A. L. Cell. Biol. PubMed Scopus Google Scholar, Wolf B.B. K. Mosser D.D. A. T. Cohen G.M. Green D.R. Cell. Biol. PubMed Scopus Google Scholar). has been shown that Hsp72 upstream of the caspase by inhibiting the release of cytochrome c from the mitochondria D. S. T. J. J. 2003; PubMed Scopus Google Scholar, D.D. Caron A.W. Bourget L. Meriin A.B. Sherman M.Y. Massie B. Mol. Cell. Biol. PubMed Scopus Google Scholar, Exp. Cell Res. PubMed Scopus Google Scholar). This inhibition of cytochrome c release be achieved by the ability of Hsp72 to prevent in response to stress T. K. S. M. Cell PubMed Scopus Google Scholar). other reports have shown that Hsp72 to inhibit cytochrome c release but by inhibiting the activation of in J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google or by of the caspase and cytosolic activity M. Wissing D. Kokholm K. Kallunki T. Egeblad M. EMBO J. 1998; 17: 6124-6134Crossref PubMed Scopus (620) Google Scholar). It has also been that Hsp72 inhibit apoptosis by the protein mouse embryo necrosis cell pathway V.L. Meriin A.B. Mosser D.D. Caron A.W. Rits S. Shifrin V.I. Sherman M.Y. J. Biol. Chem. 1997; 272: 18033-18037Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, A.B. J.A. Gabai V.L. L. S. Mosser D.D. Sherman M.Y. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). has been shown that Hsp72 to released from the the and apoptosis that from release L. S. C. E. T. M. J.M. C. G. Cell. Biol. 2001; PubMed Scopus Google Scholar). This to some of the many and of Hsp72 We that Hsp72 apoptosis in by the release of cytochrome c from the mitochondria and not through the or other in the apoptotic In the of Hsp72 to inhibit caspase activation by the is in a range of and embryo fibroblasts from and as T. S. A. PubMed Scopus Google and by Giaccia and cells in and in a Hsp72 as K.A. Giaccia A.J. Killender M. Anderson R.L. J. Biol. Chem. 1998; 273: 17147-17153Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). a by and by in a to for the and the cells once in 7 in a mouse and Hsp72 and from cells or in and nuclear morphology by as K.A. Giaccia A.J. Killender M. Anderson R.L. J. Biol. Chem. 1998; 273: 17147-17153Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). cells in and the are expressed as the of in by of cells in for cells) from cells that the stress in and in to and to the not cells to and the by then in the in and the of protein by and to Hsp72 in cells a mouse for Hsp72 or an that both mouse and Hsp72 and both from of Apaf-1 a from L. and a from of or cells in once in and once in preparation and and in of on for then for cells) of a for to including mitochondria. protein of the by the cells by in of buffer and on for for no of cytochrome c as by activated by the addition of and cytochrome c to of and for activity by the of of to the effect of Hsp72 on activity by a of the to the and for the addition of cytochrome c and or recombinant Hsp72 in or recombinant Hsp72 in or Hsp72 in against Hsp72 by for of Hsp72 or effect of on activity by the of activity in and cells the same a of the of Cell Biol. 2001; PubMed Google Scholar). Cell activated by and cytochrome c for on a and buffer to membrane and the as Cell as for to and for to including mitochondria. from the and by Cytochrome c by c release a of the of and J.A. Cell 2003; PubMed Scopus Google Scholar). and for on in and in permeabilization of cells by by cells an c an by cells to on a on a a and to a of Hsp72 Apoptosis in to and but to shown K.A. Giaccia A.J. Killender M. Anderson R.L. J. Biol. Chem. 1998; 273: 17147-17153Abstract Full Text Full Text PDF PubMed Scopus (268) Google Hsp72 protects mouse embryo fibroblasts from apoptosis by and Hsp72 did not protect from and K.A. Giaccia A.J. Killender M. Anderson R.L. J. Biol. Chem. 1998; 273: 17147-17153Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). of Hsp72 in lines Hsp72 and is the normal has been shown that Hsp72 in these cells is that K.A. Giaccia A.J. Killender M. Anderson R.L. J. Biol. Chem. 1998; 273: 17147-17153Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Hsp72 from apoptosis, not a in apoptosis, as shown by to stress the of Hsp72 Apoptosis in on addition of the caspase the of apoptotic nuclear morphology in both and that caspase activity is for apoptosis. to inhibit apoptosis also to inhibit apoptosis in response to and not inhibition of caspase activity sufficient to a for a to cell shown in By both cell lines did not to cell and to that the not the the of cells not It has been shown that inhibit apoptosis in response to cell but that the cells to by necrosis C. M. Mol. 2001; PubMed Google Scholar, C. K. A. FEBS Lett. 1998; PubMed Scopus Google Scholar). In did a to in the presence of which through the extrinsic signaling pathway that Hsp72 apoptosis in response to caspase activity in to Hsp72 also by inhibiting caspase activity to be in These that caspase activity is for apoptosis and that Hsp72 by inhibiting the ability of the cell to activate the caspase Hsp72 Cytochrome c of in an in has been that Hsp72 directly inhibiting ability to activate in response to cytochrome c and by inhibiting the of the or by the binding of to this A. L. Cell. Biol. PubMed Scopus Google Scholar, Wolf B.B. K. Mosser D.D. A. T. Cohen G.M. Green D.R. Cell. Biol. PubMed Scopus Google Scholar). that cell from be to caspase activation in response to cytochrome c and from as shown in and the addition of cytochrome c and as not activity in from This a range of cytochrome c and that caspase activity to the in the cell lines from and cells cells not In these cell lines Hsp72 is protective against various apoptotic stresses. J. K. C. J. and L. of expressed Hsp72 to inhibit caspase activation in cell in response to cytochrome c and that Hsp72 not inhibit caspase activation through the apoptosome. of Hsp72 to in Cell in the of Cytochrome c and to the of inhibition of activity in from cells not be from the A. L. Cell. Biol. PubMed Scopus Google Scholar, Wolf B.B. K. Mosser D.D. A. T. Cohen G.M. Green D.R. Cell. Biol. PubMed Scopus Google Scholar). this from cells and cytochrome in the presence or of the same recombinant Hsp72 as in the Hsp72 to inhibit caspase activation and Hsp72 from Hsp72 in of a buffer for the caspase activation addition of recombinant Hsp72 apparent an of an buffer to that in the Hsp72 in or not caspase activation to a as the recombinant Hsp72 that had been by the ability to inhibit caspase activation and recombinant Hsp72 against a to inhibit caspase activation in also inhibitory in the same buffer of Hsp72 by ability to a binding of D.R. S. A. PubMed Scopus Google Scholar). binding of to Hsp72 by that both the binding and the binding D.R. S. A. PubMed Scopus Google Scholar, S. E. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). Hsp72 not by the protein and to the not It is important to that cells of the Hsp72 in a a that has been to the presence of the in these cells Mol. Cell. Biol. 6: PubMed Scopus Google Scholar, Cell. Full Text PDF PubMed Scopus Google Scholar). cells in this cytochrome caspase A. L. Cell. Biol. PubMed Scopus Google Scholar, Wolf B.B. K. Mosser D.D. A. T. Cohen G.M. Green D.R. Cell. Biol. PubMed Scopus Google that the high of Hsp72 function of the recombinant the from which Hsp72 and not in both recombinant Hsp72 in and heat-denatured activity against a salt buffer activity of to cytochrome c and to the of E. J. 2001; PubMed Google Scholar, K. C. X.M. Cohen G.M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google an of caspase in the caspase activation by Hsp72 in in the cell shown in is an of caspase activation this Hsp72 Apaf-1 as by to the of Hsp72 Apaf-1 by due to of Hsp72 and Apaf-1 both the and the in the not these from and cells by This the binding of to proteins both to protein complexes and to their for Cell Biol. 2001; PubMed Google Scholar). Hsp72 of and cells are shown in is activated the addition of cytochrome c and and for and for Apaf-1 the of the by the same that activate In the of cytochrome Apaf-1 as a that the Hsp72 as a a and not in or Hsp72 present in some Apaf-1 or the apoptosome. Hsp72 inhibiting be that Hsp72 to in a Apaf-1 or the the and of complexes by an and the proteins on a binding of the an in Hsp72 the ability of this to It is that the addition of or inhibit the binding of Hsp72 to Apaf-1 or to the is to inhibit binding as shown in the addition of cytochrome c to sufficient to induce some by not these no to the of Hsp72 Hsp72 to Hsp72 and Apaf-1 not be by Hsp72 the of Cytochrome c in to a of to cytochrome c release from to reports that the ability of Hsp72 to inhibit apoptosis of cytochrome c release M. Wissing D. Kokholm K. Kallunki T. Egeblad M. EMBO J. 1998; 17: 6124-6134Crossref PubMed Scopus (620) Google Scholar, Exp. Cell Res. PubMed Scopus Google Scholar). of the cells a in cytochrome c release in cells but of cytochrome c release by cytochrome c release a by and J.A. Cell 2003; PubMed Scopus Google a different permeabilization of the membrane by the release of proteins from the mitochondrial proteins are the mitochondria. cells are then in and cytochrome c is by shown in of and cells a that an c this to be to the cytochrome c not In and a of cells cytochrome c Cytochrome c release by but a of cells had cytochrome c release and apoptosis not not a of cells cytochrome c due to the of cell membrane apoptosis. Hsp72 a of cytochrome c cells the cytochrome c release and apoptosis, the cells and by a and cytochrome c present in a of a mitochondrial In cells that had been different cell apparent cells to cells or a and nuclear morphology apoptosis and a of cytochrome In of cells the cells a normal and cytochrome c or apoptotic cells and cytochrome c not cells that had not been by cytochrome c that the to the mitochondria not also and and for cytochrome c In the of an apoptotic nuclear morphology the of cytochrome c from the the different the of cells Cytochrome c the in the of in to has been that Hsp72 cytochrome c release D. S. T. J. J. 2003; PubMed Scopus Google Scholar, D.D. Caron A.W. Bourget L. Meriin A.B. Sherman M.Y. Massie B. Mol. Cell. Biol. PubMed Scopus Google Scholar, Exp. Cell Res. PubMed Scopus Google but has not been this is the critical point of Hsp72 function or Hsp72 also in the apoptotic and cells for cytochrome c release and nuclear apoptosis by Hsp72 cytochrome c release in response to and but not the shown in nuclear morphology in cytochrome c release in and to be the of a nuclear apoptotic morphology not By of a of cytochrome c cytochrome c release in response to including Hsp72 directly inhibiting the activation of caspases, we be an in the of cells cytochrome c release but a normal nuclear This not the and of the cells a healthy or a apoptotic In the caspase nuclear in response to but to inhibit cytochrome c release as J. Cohen G.M. Lett. 1998; PubMed Scopus Google Scholar). In this the cells to to the that caspases for apoptotic signaling to inhibit cytochrome c to inhibit cytochrome c release in response to not Hsp72 Apoptosis by has been that Hsp72 protects cells from stress by repairing damage to the protein through we show that Hsp72 can inhibit apoptosis by and not protein damage. and proteins induce the of proteins in is as the response S. PubMed Google Scholar). to and the of Hsp72 as a of Hsp72 to be by but not by the other role of Hsp72 in protecting cells from apoptosis has been active for a of and an of the apoptotic has the by which Hsp72 apoptosis in response to cellular and extracellular signaling is of for by Hsp72 have been including cytochrome c the of the and the inhibition of V.L. Meriin A.B. Mosser D.D. Caron A.W. Rits S. Shifrin V.I. Sherman M.Y. J. Biol. Chem. 1997; 272: 18033-18037Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 11Jaattela M. Wissing D. Kokholm K. Kallunki T. Egeblad M. EMBO J. 1998; 17: 6124-6134Crossref PubMed Scopus (620) Google Scholar, A. L. Cell. Biol. PubMed Scopus Google Scholar, Wolf B.B. K. Mosser D.D. A. T. Cohen G.M. Green D.R. Cell. Biol. PubMed Scopus Google Scholar, D. S. T. J. J. 2003; PubMed Scopus Google Scholar, D.D. Caron A.W. Bourget L. Meriin A.B. Sherman M.Y. Massie B. Mol. Cell. Biol. PubMed Scopus Google Scholar, Exp. Cell Res. PubMed Scopus Google Scholar, A.B. J.A. Gabai V.L. L. S. Mosser D.D. Sherman M.Y. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). of this be due to the different to the and is that apoptotic differ the different cell lines and is also an apparent Hsp72 those Hsp72 is expressed D.D. Caron A.W. Bourget L. Denis-Larose C. Massie B. Mol. Cell. Biol. 1997; 17: 5317-5327Crossref PubMed Scopus (872) Google Scholar). of has for a to be on the of Hsp72 and for to be in other It has been that Hsp72 can protect cells from apoptosis by inhibiting the function of the a protein cytochrome and dATP. This is responsible for activating the initiator which in and the effector the that the ability of Hsp72 to protect from apoptosis is due to the inhibition of caspase This also that Hsp72 inhibit the in activation of caspases by the addition of cytochrome c and dATP. in the of we that this not the case and that cytochrome caspase activity as in from cells Hsp72 cell lines in these from and that this failure of Hsp72 to inhibit caspase activation is a this is in to that by Li J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google cells for we In an to Hsp72 and cell to but Hsp72 to the or of In this capable of complexes Hsp72 and a binding for the ability of Hsp72 to inhibit cell death in Hsp72 shown to a for cells in response to the caspase to cell a of apoptosis This failure of to protect cells from as to apoptosis, has been in and cells C. M. Mol. 2001; PubMed Google Scholar, C. K. A. FEBS Lett. 1998; PubMed Scopus Google as well as in the fibroblasts in this In to a and inhibit cytochrome c release in through the inhibition of the caspase in the extrinsic signaling extrinsic pathway to caspase activation as a of the intrinsic pathway caspase activation is a have been reports of activity in the intrinsic apoptotic pathway upstream of mitochondrial permeabilization A. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, S. J. Cell Biol. PubMed Scopus Google reports have shown that is not an of intrinsic apoptosis J. S. J. Cell Biol. PubMed Scopus Google Scholar, M. J.M. A. J. Cell Biol. PubMed Scopus Google Scholar). the of to inhibit cytochrome c release in response to that caspases not play an important role in apoptosis in cytochrome c release has in apoptosis the inhibition of by is to inhibition of the and not the cell that Hsp72 is to the inhibition of the by Hsp72 have for the of a cell. this J. S. J. Cell Biol. PubMed Scopus Google have that the of Apaf-1 from cells did not against or cytotoxic the inhibition of apoptosis. for measuring nuclear apoptosis and cytochrome c release to the fate of cells to a of apoptotic of this is the ability to simultaneously cells for a apoptotic upstream of function c and a apoptotic dependent on caspase activation It that cells that an apoptotic nuclear morphology had also cytochrome c cytochrome c release of caspase not by the caspase which nuclear Hsp72 to be an of cytochrome c release in response to and as D. S. T. J. J. 2003; PubMed Scopus Google Scholar, D.D. Caron A.W. Bourget L. Meriin A.B. Sherman M.Y. Massie B. Mol. Cell. Biol. PubMed Scopus Google Scholar, Exp. Cell Res. PubMed Scopus Google Scholar, V.L. K. Mosser D.D. Sherman M.Y. Mol. Cell. Biol. PubMed Scopus Google Scholar). Hsp72 did not inhibit cytochrome c release or apoptosis in in response to the no that Hsp72 inhibit nuclear apoptosis once cytochrome c release had Hsp72 on the to inhibit caspase or by directly inhibiting caspase then have to cells the cell morphology in the presence of c release but normal nuclear but this not the This also by the of Hsp72 to inhibit apoptosis cytochrome c release and a of apoptosis T. B. K. 2001; PubMed Scopus Google Scholar). Hsp72 to cytochrome c release these that Hsp72 apoptosis by inhibiting mitochondrial membrane permeabilization and cytochrome c no that Hsp72 caspase activation cytochrome c and the of to protect the of cells that be for cells to intracellular these we that the addition of recombinant Hsp72 to cell caspase activation by cytochrome c and as A. L. Cell. Biol. PubMed Scopus Google Scholar, Wolf B.B. K. Mosser D.D. A. T. Cohen G.M. Green D.R. Cell. Biol. PubMed Scopus Google Scholar). This effect in from both and from cells that in the It that the effect of recombinant Hsp72 different to that of Hsp72 that had been expressed that the addition of the buffer in which Hsp72 sufficient to inhibit caspase Hsp72 in in is from and is in a buffer of the are in E. J. 2001; PubMed Google Scholar, K. C. X.M. Cohen G.M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google salt is a of It also that Hsp72 that had been in a salt buffer a ability to inhibit caspase activity and that the Hsp72 preparation from to protective function as we are in own be that Li J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google have that Hsp72 in a salt buffer capable of inhibiting caspase activation in an in Hsp72 in an buffer had no the we have from in a we their cytochrome c release has been by of cells and by this on and cells to that cytochrome c release but not by different the cells and and the cytochrome c by from the of cells mitochondrial the of measuring cell can be is to and not be to in cytochrome c release this fate of cell an apoptotic is on a and this is the same that be for the of apoptosis. that Hsp72 by inhibiting cytochrome c by directly on the mitochondria or some no that Hsp72 apoptosis in once cytochrome c release had is that inhibition of the to This a to the of the anti-apoptotic function of the apoptotic pathway leading to cytochrome c release is and the by which Hsp72 this pathway It is that repairing protein damage a role in inhibiting apoptosis, signaling through extracellular receptors not protein damage and as not induce as shown in is that the ability of Hsp72 to inhibit apoptosis is and from ability to inhibit necrosis D. Li G.C. S. A. 1995; PubMed Scopus Google Scholar). from necrosis is to be achieved by the of the protein through the function of also to D. and A. for and to Killender for

The p16 (CDKN2/MTS-1/INK4A) gene is one of several tumour-suppressor genes that have been shown to be inactivated by DNA methylation in various human cancers including breast tumours. We have used bisulphite genomic sequencing to examine the detailed sequence specificity of DNA methylation in the CpG island promoter/exon 1 region in the p16 gene in DNA from a series of human breast cancer specimens and normal human breast tissue (from reductive mammaplasty). The p16 region examined was unmethylated in the four normal human breast specimens and in four out of nine breast tumours. In the other five independent breast tumour specimens, a uniform pattern of DNA methylation was observed. Of the nine major sites of DNA methylation in the amplified region from these tumour DNAs, four were in non-CG sequences. This unusual concentration of non-CG methylation sites was not a general phenomenon present throughout the genome of these tumour cells because the methylated CpG island regions of interspersed L1 repeats had a pattern of (almost exclusively) CG methylation similar to that found in normal breast tissue DNA and in DNA from tumours with unmethylated p16 genes. These data suggest that DNA methylation of the p16 gene in some breast tumours could be the result of an active process that generates a discrete methylation pattern and, hence, could ultimately be amenable to therapeutic manipulation.

Efficient recovery of clones from the 5' end of the human L1 dispersed repetitive elements necessitates the use of deletion mcr- host strains since this region contains a CpG island which is hypermethylated in vivo. Clones recovered with conventional mcr+ hosts seem to have been derived preferentially from L1 members which have accumulated mutations that have removed sites of methylation. We present a revised consensus from the 5' presumptive control region of these elements. This revised consensus contains a consensus RNA polymerase III promoter which would permit the synthesis of transcripts from the 5' end of full length L1 elements. Such potential transcripts are likely to exhibit a high degree of secondary structure. In addition, we have determined the flanking sequences for 6 full length L1 elements. The majority of full length L1 clones show no convincing evidence for target site duplication in the insertion site as commonly observed with truncated L1 elements. These data would be consistent with two mechanisms of integration of transposing L1 elements with different mechanisms predominating for full length and truncated elements.