In the analysis of the effects of radiation or drugs on clonogenic survival data of mammalian cells, it is often advantageous to compare entire dose-response curves generated under different experimental conditions rather than to conduct single-dose comparisons. We propose a two-stage method for the global comparison of such curves. The first stage consists of individual fits of a flexible model to the dose-response sequences. The second stage treats the fitted coefficients as data and analyzes them jointly using a multivariate analysis of variance. For dose-response models of the type commonly used in the analysis of clonogenic survival data, this method allows the definition of a statistical test for interaction among treatments. That is, a test that the combination of agents produces a dose-response curve which is not what would be expected if the agents were acting multiplicatively (additively in the log cell survival scale). Analyses of cell survival curves by this method for experiments on iododeoxyuridine-mediated radiosensitization and on chemosensitization of bleomycin cytotoxicity in a human bladder cancer cell line (647V) are presented.
Resistance to 5-fluorouracil (5-FU) has been associated with thymidylate synthase (TS) gene amplification and increased TS protein levels. Increased TS protein expression has also been found to be a significant independent prognostic factor for disease-free survival and overall survival in patients treated with adjuvant 5-FU-based chemotherapy. In these studies and in our prior preclinical studies, TS has been considered a marker of proliferative capacity. The purpose of the current study was to further evaluate the association between TS levels and cell cycle regulation, by investigating cell cycle kinetics in a 5-FU-resistant cell line with constitutive overexpression of TS. The influence of increased TS levels on cell cycle progression may provide insight into methods to overcome 5-FU resistance.5-FU-sensitive NCI H630(WT) and 5-FU-resistant NCI H630(R1) (with 15- to 20-fold higher TS protein levels) were utilized in this investigation to determine the influence of constitutive overexpression of TS on cell cycle kinetics.There was no apparent influence of increased TS levels on cell cycle distribution during asynchronous growth, and both cell lines reach plateau growth phase in 120 hours, arresting in G0/G1 as determined by flow cytometry. In the H630(WT) cells, this G0/ G1 arrest was associated with a 14- to 17-fold reduction in TS activity and protein levels (using the TS-106 monoclonal antibody), whereas in the H630(R1) cells, only a two- to fivefold reduction was noted. Flow cytometry analysis utilizing Ki-67 indicated that there was no evidence of a G0 population in the confluent H630(R1), whereas 26% +/- 7% of confluent H630(WT) cells were Ki-67 negative (G0) and the remainder had low Ki-67 signal intensity. Analysis of pRb phosphorylation and p16 and p21 expression suggested that the arrest point for both cell lines was before the point at which Rb phosphorylation takes place, yet the confluent H630(R1) cells had threefold higher p21 than confluent H630(WT) cells.These data suggest that the 5-FU-resistant H630(R1) cell lines arrest at a later point in G0/G1 and have a potentially greater capacity for proliferation.
Nuclear clusterin (nCLU) is an ionizing radiation (IR)-inducible protein that binds Ku70, and triggers apoptosis when overexpressed in MCF-7 cells. We demonstrate that endogenous nCLU synthesis is a product of alternative splicing. Reverse transcriptase-PCR analyses revealed that exon II, containing the first AUG and encoding the endoplasmic reticulum-targeting peptide, was omitted. Exons I and III are spliced together placing a downstream AUG in exon III as the first available translation start site. This shorter mRNA produces the 49-kDa precursor nCLU protein. Ku70 binding activity was localized to the C-terminal coiled-coil domain of nCLU. Leucine residues 357, 358, and 361 of nCLU were necessary for Ku70-nCLU interaction. The N- and C-terminal coiled-coil domains of nCLU interacted with each other, suggesting that the protein could dimerize or fold. Mutation analyses indicate that the C-terminal NLS was functional in nCLU with the same contribution from N-terminal NLS. The C-terminal coiled-coil domain of nCLU was the minimal region required for Ku binding and apoptosis. MCF-7 cells show nuclear as well as cytoplasmic expression of GFP-nCLU in apoptotic cells. Cytosolic aggregation of GFP-nCLU was found in viable cells. These results indicate that an inactive precursor of nCLU exists in the cytoplasm of non-irradiated MCF-7 cells, translocates into the nucleus following IR, and induces apoptosis. Nuclear clusterin (nCLU) is an ionizing radiation (IR)-inducible protein that binds Ku70, and triggers apoptosis when overexpressed in MCF-7 cells. We demonstrate that endogenous nCLU synthesis is a product of alternative splicing. Reverse transcriptase-PCR analyses revealed that exon II, containing the first AUG and encoding the endoplasmic reticulum-targeting peptide, was omitted. Exons I and III are spliced together placing a downstream AUG in exon III as the first available translation start site. This shorter mRNA produces the 49-kDa precursor nCLU protein. Ku70 binding activity was localized to the C-terminal coiled-coil domain of nCLU. Leucine residues 357, 358, and 361 of nCLU were necessary for Ku70-nCLU interaction. The N- and C-terminal coiled-coil domains of nCLU interacted with each other, suggesting that the protein could dimerize or fold. Mutation analyses indicate that the C-terminal NLS was functional in nCLU with the same contribution from N-terminal NLS. The C-terminal coiled-coil domain of nCLU was the minimal region required for Ku binding and apoptosis. MCF-7 cells show nuclear as well as cytoplasmic expression of GFP-nCLU in apoptotic cells. Cytosolic aggregation of GFP-nCLU was found in viable cells. These results indicate that an inactive precursor of nCLU exists in the cytoplasm of non-irradiated MCF-7 cells, translocates into the nucleus following IR, and induces apoptosis. clusterin amino acid nuclear clusterin secreted clusterin precursor of nuclear clusterin green fluorescent protein glutathione sulfotransferase MCF-7:WS8 ionizing radiation endoplasmic reticulum N-terminal domain of nCLU (aa 18–97) central region of nCLU (aa 98–317) C-terminal coiled-coil region of nCLU (aa 318–368) C-terminal non-coiled coil region of nCLU (aa 367–448) nuclear localization signal 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside reverse transcriptase endoplasmic reticulum gray The clusterin (CLU)1gene was originally isolated by numerous laboratories due to its up-regulation during cell death responses in various tissues and after various toxic stress signals (1Danik M. Chabot J.G. Mercier C. Benabid A.L. Chauvin C. Quirion R. Suh M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8577-8581Google Scholar, 2Bursch W. Gleeson T. Kleine L. Tenniswood M. Arch. Toxicol. 1995; 69: 253-258Google Scholar, 3Buttyan R. Olsson C.A. Pintar J. Chang C. Bandyk M. Ng P.Y. Sawczuk I.S. Mol. Cell. Biol. 1989; 9: 3473-3481Google Scholar, 4Ahuja H.S. Tenniswood M. Lockshin R. Zakeri Z.F. Biochem. Cell Biol. 1994; 72: 523-530Google Scholar, 5Leger J.G. Montpetit M.L. Tenniswood M.P. Biochem. Biophys. Res. Commun. 1987; 147: 196-203Google Scholar, 6Leger J.G. Le Guellec R. Tenniswood M.P. Prostate. 1988; 13: 131-142Google Scholar, 7May P.C. Ann. N. Y. Acad. Sci. 1993; 679: 235-244Google Scholar, 8Montpetit M.L. Lawless K.R. Tenniswood M. Prostate. 1986; 8: 25-36Google Scholar, 9Sensibar J.A. Sutkowski D.M. Raffo A. Buttyan R. Griswold M.D. Sylvester S.R. Kozlowski J.M. Lee C. Cancer Res. 1995; 55: 2431-2437Google Scholar, 10Tenniswood M.P. Guenette R.S. Lakins J. Mooibroek M. Wong P. Welsh J.E. Cancer Metastasis Rev. 1992; 11: 197-220Google Scholar, 11Wong P. Biochem. Cell Biol. 1994; 72: 489-498Google Scholar). CLU expression is complex, appearing as different forms in different cell compartments. One set of proteins is directed for secretion, and other CLU species are expressed in the cytoplasm and nucleus (see below). The secretory form of the clusterin protein (sCLU) has been extensively studied and is produced by translation from the first AUG codon of the full-length CLU mRNA. sCLU is targeted to the ER by an initial leader peptide. This ∼60-kDa pre-sCLU protein is further glycosylated and proteolytically cleaved into α- and β-subunits, held together by disulfide bonds (12Wong P. Pineault J. Lakins J. Taillefer D. Leger J. Wang C. Tenniswood M. J. Biol. Chem. 1993; 268: 5021-5031Google Scholar, 13Yang C.R. Leskov K. Hosley-Eberlein K. Criswell T. Pink J.J. Kinsella T.J. Boothman D.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5907-5912Google Scholar). External sCLU is an 80-kDa protein that appears as an ∼40-kDa α- and β-protein smear by SDS-PAGE under reducing conditions (14Yang C.R. Yeh S. Leskov K. Odegaard E. Hsu H.L. Chang C. Kinsella T.J. Chen D.J. Boothman D.A. Nucleic Acids Res. 1999; 27: 2165-2174Google Scholar). The role(s) of CLU in cell death processes is also complex and may be different for the various forms of the protein. Recent data suggest that overexpression of sCLU in human cancer cells caused drug resistance and protection against certain cytotoxic agents that induce apoptosis (9Sensibar J.A. Sutkowski D.M. Raffo A. Buttyan R. Griswold M.D. Sylvester S.R. Kozlowski J.M. Lee C. Cancer Res. 1995; 55: 2431-2437Google Scholar, 15Steinberg J. Oyasu R. Lang S. Sintich S. Rademaker A. Lee C. Kozlowski J.M. Sensibar J.A. Clin. Cancer Res. 1997; 3: 1707-1711Google Scholar, 16Miyake H. Nelson C. Rennie P.S. Gleave M.E. Cancer Res. 2000; 60: 2547-2554Google Scholar). Additional confirmatory data suggest that sCLU acts as a molecular chaperone, scavenging denatured proteins outside cells following specific stress-induced injury such as heat shock (17Clark A.M. Griswold M.D. J. Androl. 1997; 18: 257-263Google Scholar, 18Kimura K. Asami K. Yamamoto M. Cell Biochem. Funct. 1997; 15: 251-257Google Scholar, 19Michel D. Chatelain G. North S. Brun G. Biochem. J. 1997; 328: 45-50Google Scholar, 20Humphreys D.T. Carver J.A. Easterbrook-Smith S.B. Wilson M.R. J. Biol. Chem. 1999; 274: 6875-6881Google Scholar, 21Viard I. Wehrli P. Jornot L. Bullani R. Vechietti J.L. Schifferli J.A. Tschopp J. French L.E. J. Invest. Dermatol. 1999; 112: 290-296Google Scholar). In fact, sCLU possesses nonspecific binding activity to hydrophobic domains of various proteins in vitro (20Humphreys D.T. Carver J.A. Easterbrook-Smith S.B. Wilson M.R. J. Biol. Chem. 1999; 274: 6875-6881Google Scholar), further supporting its role as a molecular chaperone acting to clear cellular debris. sCLU does not associate with Ku70 (14Yang C.R. Yeh S. Leskov K. Odegaard E. Hsu H.L. Chang C. Kinsella T.J. Chen D.J. Boothman D.A. Nucleic Acids Res. 1999; 27: 2165-2174Google Scholar). Other data, including our own (13Yang C.R. Leskov K. Hosley-Eberlein K. Criswell T. Pink J.J. Kinsella T.J. Boothman D.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5907-5912Google Scholar), show that overexpression of a specific nuclear form of CLU (nCLU) acts as a pro-death signal, inhibiting cell growth and survival. Our laboratory isolated CLU while searching for Ku70-binding proteins using a yeast two-hybrid screen of a human liver cDNA library (14Yang C.R. Yeh S. Leskov K. Odegaard E. Hsu H.L. Chang C. Kinsella T.J. Chen D.J. Boothman D.A. Nucleic Acids Res. 1999; 27: 2165-2174Google Scholar). The specificity of nCLU-Ku70 interaction was confirmed by co-immunoprecipitation, far Western, and confocal co-localization analyses of the two proteins in transfected or IR-treated MCF-7:WS8 (MCF-7) breast adenocarcinoma cells (13Yang C.R. Leskov K. Hosley-Eberlein K. Criswell T. Pink J.J. Kinsella T.J. Boothman D.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5907-5912Google Scholar). Confocal microscopy revealed an apparently inactive form of nCLU (i.e. pnCLU) in the cytoplasm of non-irradiated cells (13Yang C.R. Leskov K. Hosley-Eberlein K. Criswell T. Pink J.J. Kinsella T.J. Boothman D.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5907-5912Google Scholar) that subsequently translocated to the nucleus following clinically relevant low dose IR exposures (≥1Gy). Co-immunoprecipitation studies demonstrated the presence of the Ku80 protein in the Ku70-nCLU complex, strongly suggesting that nCLU bound to the Ku70/Ku80 dimer (13Yang C.R. Leskov K. Hosley-Eberlein K. Criswell T. Pink J.J. Kinsella T.J. Boothman D.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5907-5912Google Scholar). Overexpression of nCLU also blocked Ku70/Ku80 DNA end binding activity (14Yang C.R. Yeh S. Leskov K. Odegaard E. Hsu H.L. Chang C. Kinsella T.J. Chen D.J. Boothman D.A. Nucleic Acids Res. 1999; 27: 2165-2174Google Scholar); however, the relationship between nCLU binding to Ku70 and cell death remained undetermined. Interestingly, an ∼49-kDa nuclear CLU precursor (pnCLU) was induced and translocated from the cytoplasm to the nucleus after certain cytotoxic events, including IR (13Yang C.R. Leskov K. Hosley-Eberlein K. Criswell T. Pink J.J. Kinsella T.J. Boothman D.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5907-5912Google Scholar) or transforming growth factor-β (22Reddy K.B. Jin G. Karode M.C. Harmony J.A. Howe P.H. Biochemistry. 1996; 35: 6157-6163Google Scholar) treatments. This "death" form of the CLU protein was proposed to be synthesized from a second in-frame AUG codon (13Yang C.R. Leskov K. Hosley-Eberlein K. Criswell T. Pink J.J. Kinsella T.J. Boothman D.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5907-5912Google Scholar, 22Reddy K.B. Jin G. Karode M.C. Harmony J.A. Howe P.H. Biochemistry. 1996; 35: 6157-6163Google Scholar), although mechanisms of production were not elucidated. The pnCLU protein apparently lacks the ER-targeting leader peptide and does not undergo α/β cleavage or extensive glycosylation, as observed with sCLU (22Reddy K.B. Jin G. Karode M.C. Harmony J.A. Howe P.H. Biochemistry. 1996; 35: 6157-6163Google Scholar). pnCLU functions remain unknown. Following IR treatment of MCF-7 cells, an ∼55-kDa nCLU protein was expressed at extremely low levels in the nuclei of exposed cells (13Yang C.R. Leskov K. Hosley-Eberlein K. Criswell T. Pink J.J. Kinsella T.J. Boothman D.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5907-5912Google Scholar). Overexpression of GFP-fused nCLU (initiated from the second AUG start codon in its mRNA) or a C-terminal 120-amino acid fragment (C-120) in MCF-7 cells resulted in significant growth inhibition, caspase 3-independent apoptosis, and lethality (13Yang C.R. Leskov K. Hosley-Eberlein K. Criswell T. Pink J.J. Kinsella T.J. Boothman D.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5907-5912Google Scholar, 23Han B.H. DeMattos R.B. Dugan L.L. Kim-Han J.S. Brendza R.P. Fryer J.D. Kierson M. Cirrito J. Quick K. Harmony J.A. Aronow B.J. Holtzman D.M. Nat. Med. 2001; 7: 338-343Google Scholar). All attempts to stably express nCLU resulted in lethality (13Yang C.R. Leskov K. Hosley-Eberlein K. Criswell T. Pink J.J. Kinsella T.J. Boothman D.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5907-5912Google Scholar) or clones expressing G418 resistance without nCLU. 2C.-R. Yang, K. S. Leskov, and D. A. Boothman, unpublished observations.2C.-R. Yang, K. S. Leskov, and D. A. Boothman, unpublished observations. In contrast, overexpression of sCLU protein did not affect the survival of MCF-7 cells before or after IR stress (14Yang C.R. Yeh S. Leskov K. Odegaard E. Hsu H.L. Chang C. Kinsella T.J. Chen D.J. Boothman D.A. Nucleic Acids Res. 1999; 27: 2165-2174Google Scholar). Analyses of the amino acid sequence of full-length rat CLU detected four "myosin tail-like" domains (24Tsuruta J.K. Wong K. Fritz I.B. Griswold M.D. Biochem. J. 1990; 268: 571-578Google Scholar) that are synonymous with a "coiled-coil" structure. Coiled-coil domains are the most common motifs used by proteins for oligomerization, and consist of amphipathic α-helices usually with a 4-3 pattern of hydrophobic and hydrophilic residues repeated in heptads. To date, no structure/function studies of the CLU protein have been performed, and mechanisms of the production of nuclear forms of this protein has yet to be determined. The objectives of the following studies were as follows: (a) investigate the mechanism(s) of nCLU production; (b) identify the functional NLS of the protein; (c) identify the death domain of nCLU, and (d) elucidate the Ku70-binding domain of nCLU. These studies may then allow elucidation of the role of nCLU-Ku70 binding in cell death responses. We demonstrate that nCLU has one high affinity coiled-coil Ku70 binding domain within its C terminus. This domain is essential for its cell death function. Furthermore, nCLU contains an additional coiled-coil domain in its N terminus that interacts with its own C-terminal coiled-coil domain. Interestingly, the N-terminal coiled-coil domain of nCLU does not interact with Ku70. Furthermore, an N-terminal nCLU coiled-coil-containing peptide interfered with binding of the nCLU C-terminal coiled-coil domain with Ku70. Based on the apparent physical interactions of N- and C-terminal pnCLU coiled-coil domains, we offer a mechanism by which the inert ∼49-kDa pnCLU could become activated to an ∼55-kDa nCLU death protein. Finally, we discuss the cell death function of nCLU in damaged cells, and we propose mechanisms of cell death involving Ku70 binding. Based on probable splice sites, the following primers were used to investigate whether alternative splice forms of CLU could be found: hCLU5′, 5′-ACAGGGTGCCGCTGACC-3′; hCLUN-term-rev, 5′-TTAGAGCTCCTTCAGCTTTGTCTC TG-3′; NCLUforw-3, 5′-GCTGACCGAAATGTC-3′; hnCLUrev, 5′-TCACACCACCCGGTGCTTTTTG-3′. Total RNA from MCF-7 cells was extracted using RNAsol B (Invitrogen). Reverse transcription reactions were performed using the oligo(dT) primer (Promega) and Superscript II enzyme system (Invitrogen). The PCR step was performed using Taq DNA polymerase (Invitrogen). When hCLU5′ and hCLUN-term-rev primers were used, PCR conditions were as follows: 95 °C for 30 s, 62–72 °C for 45 s, and 72 °C for 45 s. When NCLUforw-3 primer was used in pair with either hCLUN-term-rev or hnCLUrev primers, optimal conditions were as follows: 95 °C for 30 s, 64 °C for 45 s, and 72 °C for 45 s. A Bio-Rad iCycler Thermal Cycler was used for all reactions. RT-PCR products were resolved by 2% agarose gel electrophoresis and purified using the Concept Matrix Gel Extraction System (Invitrogen). DNA sequencing was performed by the Case Western Reserve University DNA Sequencing Service. EMBnet software (www.ch.embnet.org/software/COILS_form.html) was used to analyze amino acid (aa) sequences of mouse and human CLU proteins for coiled-coil domain structures; coiled-coil domains in proteins are known to be important for protein-protein interactions (25Alber T. Curr. Opin. Genet. & Dev. 1992; 2: 205-210Google Scholar, 26Hirai S. Yaniv M. New Biol. 1989; 1: 181-191Google Scholar, 27Kouzarides T. Packham G. Cook A. Farrell P.J. Oncogene. 1991; 6: 195-204Google Scholar). Analyses were performed using the algorithm published by Lupas et al.(28Lupas A. Van Dyke M. Stock J. Science. 1991; 252: 1162-1164Google Scholar). Primary sequences of both human and mouse CLU proteins were retrieved from GenBankTM (accession numbers: S70244 for mouse and M64722 for human CLUs (12Wong P. Pineault J. Lakins J. Taillefer D. Leger J. Wang C. Tenniswood M. J. Biol. Chem. 1993; 268: 5021-5031Google Scholar, 29Jordan-Starck T.C. Lund S.D. Witte D.P. Aronow B.J. Ley C.A. Stuart W.D. Swertfeger D.K. Clayton L.R. Sells S.F. Paigen B. J. Lipid Res. 1994; 35: 194-210Google Scholar)). Construction of PCR primer sets for deletion/mutation analyses of the CLU protein was performed using OligoTech version 1.00 software (Oligos Etc., Inc./Oligos Therapeutics, Wilsonville, OR). The Y190 yeast strain (HIS3,lacZ,trp1,leu2 (30Flick J.S. Johnston M. Mol. Cell. Biol. 1990; 10: 4757-4769Google Scholar, 31Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Google Scholar)) was used in two-hybrid structure/function analyses as described (13Yang C.R. Leskov K. Hosley-Eberlein K. Criswell T. Pink J.J. Kinsella T.J. Boothman D.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5907-5912Google Scholar, 14Yang C.R. Yeh S. Leskov K. Odegaard E. Hsu H.L. Chang C. Kinsella T.J. Chen D.J. Boothman D.A. Nucleic Acids Res. 1999; 27: 2165-2174Google Scholar). Full-length mouse CLU cDNA was a generous gift from Dr. M. Tenniswood (Notre Dame University). Full-length mouse Ku70 cDNA was a generous gift from Dr. M. Abe (National Institute of Radiological Sciences, Japan). Human CLU and Ku70 constructs were used as described previously (14Yang C.R. Yeh S. Leskov K. Odegaard E. Hsu H.L. Chang C. Kinsella T.J. Chen D.J. Boothman D.A. Nucleic Acids Res. 1999; 27: 2165-2174Google Scholar). Mouse CLU deletion mutants, containing aa residues 18–448, 18–97, 98–317, 18–380, 18–328, 256–328, 345–380, and 318–368, and human CLU deletion fragments 34–449 and 245–380 were created by PCR using the appropriate primer sets (described above). Deletion mutants were inserted into either pAS2-1 or pACT2 vectors (Clontech Laboratories, Inc., Palo Alto, CA) as prey or bait, respectively, using SmaI sites and the Perfectly BluntTM Cloning kit (Novagen, Inc., Madison, WI), according to manufacturer's instructions. Mouse CLU deletion mutants containing amino acids 256–448, 256–380, and 256–344 were created using NcoI restriction enzyme digestion in combination withBamHI, ScaI, or PstI, respectively. The resulting fragments were then inserted into the pAS2-1 vector usingNcoI, BamHI, SmaI, and PstI restriction sites. Mouse Ku70 cDNA was excised from the original vector using BspLU11I and EcoRI enzymes, and inserted into pACT2 and pAS2-1 vectors, which were pre-digested withNcoI and EcoRI restriction enzymes. Point mutations within mouse CLU cDNAs were introduced using the QuikChange kit (Stratagene, Cedar Creek, TX) following the manufacturer's instructions. The LR mutant (substitution of 9 Leu residues to Arg within the C-terminal coiled-coil domain of mouse nCLU) was generated by direct cloning of a 155-bp DNA fragment containing all required nucleotide substitutions. This was accomplished by first synthesizing a single strand DNA fragment: 5′ GC CAG GAG CGG AAC GAC TCG CGC CAG GTG GCC GAG AGG CGGACA GAG CAG TAC AAG GAG CGG CGG CAG TCC TTC CAG TCG AAGATG CGC AAC ACC TCA TCC CGG CGG GAG CAG CGG AAC GAC CAGTTC AAC TGG GTG TCC CAG CGG GCT AAC CGC A 3′. This fragment was further amplified by PCR using Pfu DNA polymerase (Stratagene, Cedar Creek, TX) and the following pair of primers: AlmutPCR1 (30-mer), 5′ GCT AAC CTG CGC CAG GAG CGG AAC GAC TCG 3′, and AlmutPCR2 (38-mer), 5′ TTA GTA GTA CTT GTC TTC TCC CTG TGT GCG GTT AGC CC 3′. The PCR product was gel-purified, digested with the EcoNI restriction enzyme, and inserted into the pASnCLU, pAS-(256–448) and pGFPnCLU nCLU vectors, which were pre-digested with EcoNI to create NCLU-LR, NcoLR, and GFP-NCLU-LR, respectively. In addition, the undigested PCR product was inserted into a pcDNA3.1/NT-GFP-TOPO mammalian expression vector (Invitrogen) using blunt ligation to create the GFP-CcLR mutant. Human CLU cDNA fragments encoding aa residues 84–449 and 329–449 in the pACT2 vector, as well as human Ku70 cDNA in the pAS2 vector, were described previously (13Yang C.R. Leskov K. Hosley-Eberlein K. Criswell T. Pink J.J. Kinsella T.J. Boothman D.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5907-5912Google Scholar, 14Yang C.R. Yeh S. Leskov K. Odegaard E. Hsu H.L. Chang C. Kinsella T.J. Chen D.J. Boothman D.A. Nucleic Acids Res. 1999; 27: 2165-2174Google Scholar). All constructs were sequenced to verify in-frame positions with respect to the GAL4 domain. GAL4-Ku70 was co-transfected with various GAL4-CLU fragments into Y190 yeast using the protocol provided in the MATCHMAKER (Clontech Laboratories, Inc., Palo Alto, CA) manual. Stable transformants were grown for 6 days at 30 °C on plates containing His(−), Trp(−), Leu(−) and SD medium (Difco) containing 2% agarose and 15 mm 3-amino-1,2,4-triazole. Colonies were subjected to on-filter β-galactosidase assays, with X-gal as a substrate. The reaction was considered positive when colonies turned blue within first 120 min of incubation with X-gal. Negative reactions (i.e. blue colonies were not observed) were indicative of no association, only when protein expression for each CLU or Ku70 mutant could be demonstrated by Western blot analyses (see below). Blue colonies that appeared after 120 min were regarded as false-positive signals. X-gal staining was performed on colony-lift filters as described (14Yang C.R. Yeh S. Leskov K. Odegaard E. Hsu H.L. Chang C. Kinsella T.J. Chen D.J. Boothman D.A. Nucleic Acids Res. 1999; 27: 2165-2174Google Scholar). Rabbit polyclonal H-330 antibody (Santa Cruz Biotechnology) was used to detect the 49-kDa species of CLU (pnCLU) in whole cell extracts from MCF-7 cells. A 1:2500 dilution of primary H-330 antibody was used followed by 1:10,000 dilution of horseradish peroxidase-conjugated anti-rabbit secondary antibody (Santa Cruz Biotechnology). Yeast extracts were prepared using acid-washed glass beads as described in the MATCHMAKER kit manual and were used for Western blot analyses. Protein concentrations were determined using the Bio-Rad protein concentration reagent as per manufacturer's instructions (Bio-Rad). Yeast extracts were mixed with an equal volume of 2× Laemmli buffer, boiled for 5 min, and resolved on either 10 or 15% PAGE gels, depending on the expected molecular weight of the proteins of interest. Proteins were then transferred onto polyvinylidene difluoride-Plus membranes (MSI, Westboro, MA). Membranes were blocked with 5% non-fat dried milk and probed with anti-GAL4BD or anti-GAL4AD monoclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Horseradish peroxidase-conjugated anti-mouse secondary antibody (Amersham Biosciences) was then used, and proteins were visualized by ECL (Amersham Biosciences) and x-ray film. All CLU deletion or point mutants were approximately equally expressed in yeast, and no loss of stability of proteins was noted. Full-length mouse Ku70 cDNA was amplified using PCR and the appropriate primer sets and inserted into the pGEX-2T vector (Amersham Biosciences) at aSmaI restriction site to generate an in-frame glutathioneS-transferase (GST)-fused protein. Escherichia coli NovaBlue Single Competent Cells (Novagen, Inc., Madison, WI) were used for plasmid propagation according to the protocol described in the Perfectly BluntTM Cloning kit manual (Novagen, Inc., Madison, WI). GST or GST-Ku70 fusion proteins were generated and purified using standard methods (32Acharya S. Wilson T. Gradia S. Kane M.F. Guerrette S. Marsischky G.T. Kolodner R. Fishel R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13629-13634Google Scholar). All genetically manipulated plasmids were confirmed by DNA sequencing prior to use. Glutathione-agarose beads (Amersham Biosciences) were used for purification of GST-fused proteins as described (32Acharya S. Wilson T. Gradia S. Kane M.F. Guerrette S. Marsischky G.T. Kolodner R. Fishel R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13629-13634Google Scholar) using Binding buffer (20 mm Tris-HCl, pH 7.2, 150 mm NaCl, 1.5 g/liter bovine serum albumin, proteinase inhibitors). Amounts of immobilized GST-Ku70 or GST (alone) proteins were estimated by Coomassie Blue staining of SDS-PAGE gels, with various amounts of bovine serum albumin protein used as standards. Mouse CLU cDNA fragments encoding amino acid residues 18–448 and 345–380 were amplified by PCR and inserted into the pT7Blue-2 vector (Novagen, Inc., Madison, WI) using the Perfectly BluntTM Cloning kit (Novagen, Inc., Madison, WI) according to manufacturer's instructions. All cDNA expression vectors were sequenced to confirm cloning before use. The TnT-coupled wheat germ extract system (Promega, Madison, WI) was used to generate S·Tag-fused,35S-labeled CLU protein fragments in vitroaccording to manufacturer's protocols. The amount of35S-labeled protein was estimated using the S·Tag Rapid Assay kit (Novagen Inc., Madison, WI), and an equal amount of transcription/translation mixture (40 μl) was added to glutathione-containing beads at the same molar amounts (0.2 pmol of CLU to 0.2 pmol of GST or GST-Ku70) and incubated overnight at room temperature. The mixture was then incubated at 4, 23, or 37 °C for 1 h, and beads were collected by centrifugation (15,000 ×g), washed three times with ice-cold Binding buffer, resuspended in Laemmli buffer, and boiled, and proteins were resolved by 10–15% SDS-PAGE. 35S-Labeled nCLU fragments were then visualized by autoradiography as described (33Boothman D.A. Bouvard I. Hughes E.N. Cancer Res. 1989; 49: 2871-2878Google Scholar, 34van Straaten F. Muller R. Curran T. Van Beveren C. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3183-3187Google Scholar, 35Hope I.A. Struhl K. Cell. 1985; 43: 177-188Google Scholar, 36Bohmann D. Bos T.J. Admon A. Nishimura T. Vogt P.K. Tjian R. Science. 1987; 238: 1386-1392Google Scholar, 37Biggin M. Bodescot M. Perricaudet M. Farrell P. J. Virol. 1987; 61: 3120-3132Google Scholar). To study the intracellular targeting of various nCLU fragments, we transfected MCF-7 cells with GFP-nCLU and GFP-nCLU deletion mutants. Effectin reagent (Qiagen Inc., Valencia, CA) was used to transfect 2 × 104 log phase MCF-7 cells that were pre-attached for 24 h to coverslips. Forty eight hours (48 h) later, cells were fixed for 1 h in freshly prepared 2% paraformaldehyde at room temperature. Cells were then rinsed twice in phosphate-buffered saline and treated with RNase A in phosphate-buffered saline (1 μg/ml) for 20 min at 37 °C. Cells were mounted in propidium iodide-containing Vectashield mounting media (Vector Laboratories Inc., Burlingame, CA) and subjected to confocal microscopy using a Bio-Rad MRC 1024 Confocal Laser Scanning microscope (Bio-Rad). All photomicrographs show representative z-sections at ×1000 magnification. At least 180 transfected cells (green) were visually screened for distribution of GFP-CLU protein in the cell and its co-localization with propidium iodide-stained nuclei (red). The number of cells with pycnotic propidium iodide versus normal nuclear morphology was manually counted, and percentages of apoptotic cells ± S.E. were calculated (see Table I). Likewise, the number of cells with nuclear only, cytoplasmic and nuclear, or cytoplasmic only localization of the GFP-fused proteins was manually counted. Percentages of cells with nuclear (or cytoplasmic and nuclear) or cytoplasmic only expression ± S.E. were calculated (Table II).Table IInduction of apoptosis by exogenously expressed GFP-fused nCLU protein fragmentsProtein expressedCells with pycnotic nuclei (% of total) ± S.E.GFP4 ± 1GFP-nCLU25 ± 5GFP-N-term5 ± 12GFP-Center5 ± 1GFP-Ccoil31 ± 4GFP-End2 ± 0.5GFP-nCLU L343P2 ± 0.3Log phase MCF-7 cells were transfected with either GFP- or GFP-nCLU-fused cDNAs and analyzed 48 h later by confocal microscopy. z-sections were then analyzed for protein localization (Fig. 7), and percentages of apoptotic cells among GFP-positive cells were quantified. Apoptotic cells were defined as cells with condensed and fragmented pycnotic nuclei (Fig. 7). Values are mean ± S.E., representing three independent transient transfections. At least 200 GFP-positive cells were counted for each GFP construct per experiment. Open table in a new tab Table IIMutations in NLS1 or NLS2 result in cytoplasmic localization of GFP-nCLU constructsProtein expressed% localization ± S.E.CytoplasmNucleus (nucleus and cytoplasm)GFP-Nterm wild-type43 ± 557 ± 5GFP-Nterm NLS1 mutant79 ± 321 ± 3 (2-fold)GFP-End wild-type2 ± 0.298 ± 0.2 (2-fold)GFP-End NLS2 mutant78 ± 522 ± 5GFP88 ± 212 ± 2Log phase MCF-7 cells were transfected with either GFP- or GFP-nCLU-fused cDNAs with or without mutations in NLS1 and NLS2, as described in the text, and analyzed 48 h later by confocal microscopy. z-sections were then analyzed for protein localization (Fig. 8), and the percentages of cells with cytoplasmic or cytoplasmic and nuclear localization of GFP-fused proteins were quantified. The numbers with standard errors are the result of three independent transient transfections. At least 200 GFP-positive cells were counted for each GFP construct per experiment. Open table in a new tab Log phase MCF-7 cells were transfected with either GFP- or GFP-nCLU-fused cDNAs and analyzed 48 h later by confocal microscopy. z-sections were then analyzed for protein localization (Fig. 7), and percentages of apoptotic cells among GFP-positive cells were quantified. Apoptotic cells were defined as cells with condensed and fragmented pycnotic nuclei (Fig. 7). Values are mean ± S.E., representing three independent transient transfections. At least 200 GFP-positive cells were counted for each GFP construct per experiment. Log
Photodynamic therapy (PDT) for cutaneous malignancies has been found to be an effective treatment with a range of photosensitizers. The phthalocyanine Pc 4 was developed initially for PDT of primary or metastatic cancers in the skin. A Phase I trial was initiated to evaluate the safety and pharmacokinetic profiles of systemically administered Pc 4 followed by red light (Pc 4-PDT) in cutaneous malignancies. A dose-escalation study of Pc 4 (starting dose 0.135 mg/m2) at a fixed light fluence (135 J/cm2 of 675-nm light) was initiated in patients with primary or metastatic cutaneous malignancies with the aim of establishing the maximum tolerated dose (MTD). Blood samples were taken at intervals over the first 60 hours post-PDT for pharmacokinetic analysis, and patients were evaluated for toxicity and tumor response. A total of 3 patients (2 females with breast cancer and 1 male with cutaneous lymphoma) were enrolled and treated over the dose range of 0.135 mg/m2 (first dose level) to 0.54 mg/m2 (third dose level). Grade 3 erythema within the photoirradiated area was induced in patient 2, and transient tumor regression in patient 3, in spite of the low photosensitizer doses. Pharmacokinetic observations fit a 3-compartment exponential elimination model with an initial rapid distribution phase (~0.2 hrs) and relatively long terminal elimination phase (~28 hrs), Because of restrictive exclusion criteria and resultant poor accrual, the trial was closed before MTD could be reached. While the limited accrual to this initial Phase I study did not establish the MTD nor establish a complete pharmacokinetic and safety profile of intravenous Pc 4-PDT, these preliminary data support further Phase I testing of this new photosensitizer.
Three patients with extensive liver metastases from hormone-secreting tumors were treated with external beam radiation therapy to palliate signs and symptoms of tumor mass and/or hormone secretion. These patients experienced an objective response of 3, 14, and 24 months duration, respectively, as measured by plasma hormone levels and/or computed tomography (CT) scanning. Using conventional fractionation, a dose of 2400 to 3000 rad was delivered without significant acute or late toxicity. Although these tumors have a long natural history (many years), even after the development of liver metastases, radiation therapy can provide effective palliation and should be considered as a therapeutic option.
Encouraging results continue to emerge with the use of chemoradiotherapy. This review of the current literature demonstrates some of the advances that have been achieved in a variety of common malignancies with strategies based on well-defined rationales. Preliminary randomized trials now reveal a survival advantage for chemoradiotherapy in patients with inoperable non-small cell lung cancer and patients with rectal cancer at high risk for recurrence following resection. Other studies report that concurrent chemoradiotherapy may improve local control in patients with limited-stage, small cell lung cancer or esophageal malignancies. The additional toxicity of these approaches is discussed.
