Detection of herpes simplex virus thymidine kinase polypeptides in cells labeled with 35S-methionine.

To investigate the size of herpes simplex virus (HSV) thymidine kinase (TK) polypeptides, procedures have been devised to purify the enzyme from infected cells labeled with 35S-methionine by (i) affinity chromatography on Sepharose-5'-amino-5'-deoxythymidine; (ii) preparative isoelectric focusing or preparative PAGE; and (iii) glycerol gradient centrifugation. Portions of enzyme fractions, at each purification step, were also treated with an immunoadsorbent, Sepharose-anti-HSV-1 TK immunoglobulin (IgG). Labeled polypeptides eluted from the immunoadsorbent were analyzed by electrophoresis in SDS slab gels and autoradiography. The results demonstrate that the molecular weights of HSV TK polypeptides are about 40,000. TK-negative HSV-1 mutant B2006 failed to induce the 40 K dalton polypeptide.     

间充质干细胞靶向转运胸苷激酶基因联合更昔洛韦杀伤鼻咽癌细胞

背景：研究证实,间充质干细胞能通过基因修饰成为肿瘤治疗的靶向载体。目的：观察转染胸苷激酶基因的胸苷激酶-间充质干细胞联合更昔洛韦对鼻咽癌细胞的靶向杀伤作用。方法：应用LipofectamineTM2000将表达胸苷激酶基因的重组质粒pGL3-EGFP-胸苷激酶转染至SD大鼠间充质干细胞,观察其增殖能力。应用Transwell小室观察胸苷激酶-间充质干细胞的归巢特性;将胸苷激酶-间充质干细胞植入裸鼠,观察其致瘤性;用胸苷激酶-间充质干细胞/更昔洛韦干预人鼻咽癌细胞5-8F,观察其对细胞的杀伤作用及旁观者效应。结果与结论：荧光显微镜观察及RT-PCR检测结果提示实验成功将胸苷激酶基因转染至间充质干细胞,CCK-8检测结果显示胸苷激酶-间充质干细胞与间充质干细胞增殖能力差异无显著性意义（P〉0.05）。Transwell小室迁移实验显示胸苷激酶-间充质干细胞具有归巢特性,裸鼠移植瘤实验显示胸苷激酶-间充质干细胞无致瘤性。CCK-8检测检测发现胸苷激酶-间充质干细胞/更昔洛韦具有旁观者效应,可明显降低5-8F的生存率（P〈0.01）。提示胸苷激酶-间充质干细胞/更昔洛韦对鼻咽癌细胞具有靶向迁移及杀伤作用,间充质干细胞可作为治疗鼻咽癌的理想靶向转运载体。     

Clinical-scale selection of anti-CD3/CD28-activated T cells after transduction with a retroviral vector expressing herpes simplex virus thymidine kinase and truncated nerve growth factor receptor

Activation of T cells is necessary for efficient retroviral-mediated gene transfer. In addition, if the population of infused cells is to be limited to transduced cells, a means of positive selection is required. We describe a clinical scale procedure for activation of donor T cells with anti-CD3/CD28 beads followed by transduction with a retroviral construct expressing the herpes simplex virus thymidine kinase (HSV-tk) and human nerve growth factor receptor (NGFR). Optimization of transduction parameters was performed, testing the timing of transduction, centrifugation, and the use of serum. In large-scale experiments, 3-5 x 10(8) peripheral blood mononuclear cells (PBMC) were activated with anti-CD3/CD28 beads and expanded to day 13. Transduction was accomplished using MFG-TKiNG supernatant produced from the PG13 packaging line 48 hr after T-cell activation. The mean transduction frequency was 37.5% based on NGFR expression, and the mean expansion observed was 42.6-fold (mean final cell number 1.85 x 10(10)). A comparison of the ability of the Baxter Isolex 300i and the Miltenyi CliniMACS to perform purification of NGFR+ cells suggests that greater purity can be achieved with the CliniMACS device (67.4% vs. 97.7%), while the yield of transduced cells appears higher with the Isolex 300i (41.3% vs. 23.5%). We conclude that a strategy based on activation of human T cells with anti-CD3/CD28 beads can result in sufficient transduction, expansion, and purification based on NGFR expression for clinical trials.     

Demonstration of viral thymidine kinase inhibitor and its effect on deoxynucleotide metabolism in cells infected with herpes simplex virus.

The thymidine analog 5'-ethynylthymidine was a potent inhibitor of herpes simplex virus type 1 (strain KOS)-induced thymidine kinase with a Ki value of 0.09 microM. 5'-Ethynylthymidine was less inhibitory against herpes simplex virus type 2 (strain 333)-induced thymidine kinase with a Ki of 0.38 microM and showed no inhibition against human cytosolic thymidine kinase under the conditions tested. The compound was effective against the altered thymidine kinase induced by acyclovir- and bromovinyldeoxyuridine-resistant virus variants. At 100 microM 5'-ethynylthymidine, the cellular pool size of dTTP in herpes simplex virus type 1-infected cells was 5% that of infected cells receiving no drug treatment, while there was no significant effect on the pool sizes of dATP, dGTP, and dCTP. There was a positive correlation between dTTP pools and the intracellular thymidine kinase activity of herpes simplex virus type 1-infected cells. When tested alone, 5'-ethynylthymidine exhibited no antiviral activity, but it antagonized the antiviral efficacy of five compounds which require viral thymidine kinase for their action.     

Pronounced cytostatic activity and bystander effect of a novel series of fluorescent tricyclic acyclovir and ganciclovir derivatives in herpes simplex virus thymidine kinase gene-transduced tumor cell lines

A number of tricyclic acyclovir (ACV) and ganciclovir (GCV) derivatives substituted with bulky lipophilic groups have been identified as potent and highly selective cytostatic agents against herpes simplex virus type 1 (HSV-1)-thymidine kinase (TK) gene-transduced human osteosarcoma and murine mammary carcinoma tumor cells. Although their affinity for HSV-1 TK was inferior to that of ACV or GCV, their cytostatic potency and selectivity was at least as high as observed for the parental ACV and GCV compounds. The tricyclic ACV and GCV derivatives were also endowed with a very pronounced bystander effect in cell culture, albeit at relatively high drug concentrations. Unlike ACV and GCV, the tricyclic purine derivatives are endowed with intrinsically strong fluorescent properties, which allow simple and sensitive monitoring of drug concentrations in biological fluids and tissues. Also, the lipophilicity of the tricyclic purine derivatives is much higher than that of ACV and GCV, and this may allow better uptake of these derivatives from the blood into the central nervous system.     

Inclusion of the herpes simplex thymidine kinase gene in a replicating adenovirus does not augment antitumor efficacy.

Replication-incompetent adenoviruses (Ad) carrying the herpes simplex thymidine kinase (HSVtk) gene have been used in a number of human cancer gene therapy trials, however transduction has generally been limited to a small minority of tumor cells. To solve this problem, replication-competent adenoviral vectors carrying transgenes such as HSVtk have been developed. However, contradictory evidence exists regarding the efficacy of these new vectors. Accordingly, we constructed and tested a replication-competent E3-deleted adenoviral vector containing the HSVtk suicide gene driven by the endogenous E3 promoter (Ad.wt.tk). This virus showed high level production of the HSVtk transgene and was more efficacious than a non-replicating virus in vitro, after injection into flank tumors, and against established intraperitoneal tumors. However, addition of ganciclovir (GCV) therapy to cells or tumor-bearing animals treated with the replicating vector containing the HSVtk suicide gene did not result in increased cell killing. Our results indicate that addition of HSVtk to a replicating Ad virus will not likely be useful in augmenting antitumor effects.     

Enhanced ganciclovir killing and bystander effect of human tumor cells transduced with a retroviral vector carrying a herpes simplex virus thymidine kinase gene mutant

Gene transfer of the herpes simplex virus thymidine kinase (TK) gene associated with ganciclovir (GCV) treatment can lead to death of TK-expressing cells, and of neighboring TK- cells because of the bystander effect. Thus, a small proportion of TK+ cells in a tumor can lead to its complete regression after GCV treatment. However, a lack of efficacy of gene transfer into tumors associated with low GCV sensitivity and poor bystander effect of human cancer cells currently limit the clinical use of this suicide gene therapy approach. To increase the potency of suicide gene therapy, we have tested the GCV sensitivity and the bystander effect of TK mutants that have been previously described. After retroviral transduction of the TK mutants into human tumor cells of various origins, we have found a strong killing effect of GCV with cells expressing the mutants TK30 or TKF161C. The GCV sensitivity of several human tumor cell types expressing TK30 was 9- to 500-fold higher than cells containing wild-type TK. Furthermore, TK30-expressing cells were able to kill bystander cells much more efficiently than TK-expressing cells. Thus, TK30 mutant is a promising candidate for suicide gene therapy clinical trials.     

Development of a competitive PCR method for in vitro and in vivo quantification of herpes simplex virus thymidine kinase and neomycin resistance-expressing cells used in a clinical trial

The aim of this study was to set up a sensitive and specific method to quantify the number of gene-modified cells in a gene therapy clinical trial currently underway at our institution. This trial involves the use of retrovirally transduced allogeneic T cells expressing the herpes simplex-1 thymidine kinase (HSV-TK) and neomycin-phosphotransferase (NeoR) resistance gene. Quantification by competitive PCR was performed, with two homologous internal standards (deltaTK, deltaNeoR), 30 bp shorter than the target sequences (TK, NeoR), coupled to fluorescent laser-based detection. Assessment of the amplification systems procedures was carried out for each sequence. The 30-bp deletion did not affect the amplification efficiency significantly. Determination of the plateau phase of both amplified sequences demonstrated that each sample must be quantified during the predetermined exponential phase. Finally, a blinded study of a transduced cell dilutions panel validated the overall methodology. The competitive PCR was applied to quantification of the retroviral transduction process by quantifying the NeoR gene in transduced PBMC samples (prior to G418 selection) from 18 donors in our clinical trial. A mean transduction efficiency of 9.78% +/- 1.37% was observed. We also quantified TK-expressing donor transgenic T cells in a murine GvHD model. Results demonstrated on initial expansion of donor HSV-TK- expression T cells as well as a significant ganciclovir (GCV)-induced decrease correlated with the number of circulating gene-modified T cells. Therefore, we have developed an efficient gene quantification tool that should be useful for in vivo monitoring of gene-modified cells.     

Nucleotide sequence changes in thymidine kinase gene of herpes simplex virus type 2 clones from an isolate of a patient treated with acyclovir.

To identify the nucleotide changes that occur in drug-induced thymidine kinase (TK) mutants of herpes simplex virus type 2 (HSV-2), we compared the nucleotide sequences of the tk genes of two mutant HSV-2 clones isolated from a patient who had been treated with acyclovir [9-(2-hydroxyethoxymethyl)guanine; ACV] with the nucleotide sequence of the parental TK+ HSV-2(8703) strain isolated from the same patient. One of the mutants, TK-altered (TKA) HSV-2(9637), was ACV resistant but induced the incorporation of [14C]thymidine into the DNA of infected rabbit skin cells. The nucleotide sequence of the tk gene of mutant TKA HSV-2(9637) had a single change (G to A) at nucleotide 668, which would cause an arginine-to-histidine substitution at amino acid residue 223 of the TK polypeptide. The second ACV-resistant mutant, TK- HSV-2(8710), did not induce detectable incorporation of [14C]thymidine into the DNA of infected rabbit skin cells. This mutant exhibited a deletion of a single base at nucleotide 217 of its nucleotide sequence. This deletion would cause a frameshift mutation at amino acid residue 73 and chain termination at amino acid residue 86 of the TK polypeptide. The nucleotide sequence of TK+ HSV-2(8703) was the same as that of the laboratory strain, TK+ HSV-2(333). The nucleotide sequence of a bromodeoxyuridine-resistant TK- HSV-2(333) mutant of TK+ HSV-2(333) also exhibited a single-base deletion, but at nucleotide 439. This deletion would cause a frameshift mutation at amino acid residue 147 and chain termination at amino acid residue 182. The frameshift mutations of TK- HSV(8710) and TK- HSV-2(333), respectively, occurred in sequences in which C was repeated three times and G was repeated seven times. The results raise the possibility that TK- frameshift mutations of HSV-2 may be common.     

Sustained release of low-dose ganciclovir from a silicone formulation prolonged the survival of rats with gliosarcomas under herpes simplex virus thymidine kinase suicide gene therapy

A silicone formulation of ganciclovir (GCV-pellet) was developed to enhance the cytotoxic effects of herpes simplex virus thymidine kinase suicide gene therapy. The effectiveness of this drug delivery system was assessed in a rat 9L gliosarcoma model. The GCV-pellets (1 mm in length and in diameter) used in this experiment contained a total amount of 0.15 mg of GCV. In vitro experiments demonstrated that GCV was gradually released over a period of 7 days. Five days after stereotactic tumor inoculation into the right caudate nucleus, a herpes simplex virus type 1 (HSV-1) vector expressing herpes simplex virus thymidine kinase (HSV-tk) (T1, 2x10(6) pfu) was administered at the same location. The survival rate of the group treated with the GCV-pellet was compared with that of the T1 group injected intraperitoneally (IP) with GCV (30 mg/kg/day for 7 days). The GCV-pellet-treated group had a significantly prolonged survival (a median of more than 80 days) compared with the GCV IP group (a median of 65 days) and with control groups (P<0.05). The control groups (untreated or receiving only the virus vector) had a survival of 35-38 days. The survival rate of the GCV-pellet group over 80 days was 75%, and all the rats that survived more than 80 days and did not show tumors upon histological examination of the brain were deemed cured. No toxic effects or immunological reactions were observed histologically around the pellet in brain sections from the rats treated with the GCV-pellet. After GCV-pellet inoculation into the tumor, drug concentrations were kept at 1-10 microg/g tissue for 3-4 days. When the same dose of GCV (0.15 mg) in aqueous solution was injected into the tumor, GCV concentrations reached a peak of 0.5 mg/g tissue after 30 min and decreased below measurable level within 12 h. After IP injections of 3 mg GCV, GCV concentrations in the tumor reached a peak of 5.7 microg/g tissue after 30 min and also decreased below measurable level within 12 h. This sustained release of a low and effective GCV dose with the silicone formulation significantly prolonged survival in combinations with HSV-tk expression if compared to IP administration of GCV. Histological examination suggests that the treatment appears to be safe.     

Nucleoside metabolism in herpes simplex virus-infected cells following treatment with interferon and acyclovir, a possible mechanism of synergistic antiviral activity.

Alpha interferon (IFN-alpha) and nucleoside analogs have been shown to have synergistic antiherpesvirus activity in cultured cells. The mechanisms responsible for this synergistic activity are not known, but we hypothesize that IFN-alpha-induced alterations of nucleoside metabolism in virus-infected cells may play an important role. Infection of cells with herpes simplex virus type 1 (HSV-1) led to an increase in uptake of thymidine into cells. Treatment of infected cells with recombinant IFN-alpha for 24 h prior to infection resulted in a significant reduction in the uptake of exogenous thymidine but did not reduce the apparent incorporation of exogenous thymidine into DNA. The amount of exogenous thymidine phosphorylated relative to the amount taken up was the same in IFN-alpha-treated and control cultures. IFN-alpha treatment of HSV-1-infected cells also resulted in a reduction in the pool sizes of endogenous deoxyribonucleoside-5'-triphosphates relative to those of untreated HSV-1-infected cultures. Although IFN-alpha affected the metabolism of natural nucleosides in HSV-1-infected cells, it did not significantly reduce the uptake of the antiviral guanosine analog acyclovir into HSV-1-infected cells or the amount of acyclovir-5'-triphosphate accumulated. Therefore, in IFN-alpha-treated cells the concentration of a natural nucleoside, thymidine, was reduced, as were the pools of all deoxyribonucleoside-5'-triphosphates. No decrease in acyclovir or acyclovir-5'-triphosphate concentration was observed, however, when acyclovir-treated cells were exposed to IFN-alpha. These data suggest that IFN-alpha-induced alterations in nucleoside metabolism may be one mechanism whereby IFN-alpha and acyclovir express synergistic antiherpes-virus activity.     

Identification of potential biomarkers for measuring inhibition of Src kinase activity in colon cancer cells following treatment with dasatinib

Elevated levels of Src kinase expression have been found in a variety of human epithelial cancers. Most notably in colon cancer, elevated Src expression correlates with malignant potential and is also associated with metastatic disease. Dasatinib (BMS-354825) is a novel, orally active, multi-targeted kinase inhibitor that targets Src family kinases and is currently under clinical evaluation for the treatment of solid tumors. However, the effects of dasatinib on epithelial tumors are not fully understood. We show that concentrations of dasatinib that inhibit Src activity do not inhibit proliferation in 10 of 12 colon cancer cells lines. However, inhibition of integrin-dependent adhesion and migration by dasatinib correlated with inhibition of Src activity, suggesting that dasatinib may have anti-invasive or anti-metastatic activity and antiproliferative activity in epithelial tumors. Using phospho-specific antibodies, we show that inhibition of Src activity in colon cancer cell lines correlates with reduced phosphorylation of focal adhesion kinase and paxillin on specific Src-dependent phosphorylation sites. We have validated the use of phospho-specific antibodies against Src Tyr(419) and paxillin Tyr(118) as biomarkers of dasatinib activity in vivo. Colon carcinoma-bearing mice treated with dasatinib showed a decrease in both phospho-Src Tyr(419) and phospho-paxillin Tyr(118) in peripheral blood mononuclear cells, which correlated with inhibition of Src activity in the colon tumors. Thus, peripheral blood mononuclear cells may provide a useful surrogate tissue for biomarker studies with dasatinib using inhibition of Src Tyr(419) and paxillin Tyr(118) phosphorylation as read-outs of Src activity.     

Involvement of Fas (CD95/APO-1) and Fas ligand in apoptosis induced by ganciclovir treatment of tumor cells transduced with herpes simplex virus thymidine kinase.

Transduction of cancer cells with herpes simplex virus thymidine kinase gene (HSVtk) followed by prodrug ganciclovir (GCV) treatment has been shown to induce apoptosis. In this study, four murine tumors including B16F10 melanoma, NG4TL4 sarcoma, H6 hepatoma and 1MEA 7R.1 hepatoma were found to vary in sensitivity to this gene therapy strategy in vitro but, at effective doses of GCV, the HSVtk-transduced cells of all four tumors showed similar kinetics of early rise in p53 protein levels, then cell cycle S-/G2-phase arrest and finally signs of apoptosis. Immunoblot analyses revealed that Fas (CD95/APO-1), Fas ligand (FasL) and two downstream mediators, RIP and caspase-3, (CPP32, YAMA, Apopain) were increased in GCV-treated HSVtk-transduced tumor cells the cell cycle arrest and before apoptosis. Increased expression of FasL could also be observed in vivo in HSVtk-transduced tumors induced to regress by GCV treatment. Enzyme measurements using specific substrate showed that the caspase-3 activation followed kinetically the FasL expression. More than half of the HSVtk/GCV-induced cell death could be abrogated by addition to the cell culture medium of a specific antisense oligonucleotide to block FasL synthesis, a recombinant Fas/Fc chimeric protein to compete with Fas receptor for FasL binding, or cell-permeable specific tetrapeptide inhibitors of caspase-3 or caspase-8.     

Protoapigenone, a novel flavonoid, induces apoptosis in human prostate cancer cells through activation of p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase 1/2

In this study, we investigated the anticancer effect of protoapigenone on human prostate cancer cells. Protoapigenone inhibited cell growth through arresting cancer cells at S and G(2)/M phases as well as inducing apoptosis. Blockade of cell cycle by protoapigenone was associated with an increase in the levels of inactivated phospho (p)-Cdc25C (Ser216) and a decrease in the levels of activated p-cyclin B1 (Ser147), cyclin B1, and cyclin-dependent kinase (Cdk) 2. Protoapigenone triggered apoptosis by increasing the levels of cleaved poly(ADP-ribose) polymerase and caspase-3. In addition, activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK)1/2 was a critical mediator in protoapigenone-induced cell death. Inhibition of the expression of p38 MAPK and JNK1/2 by pharmacological inhibitors or specific small interfering RNA reversed the protoapigenone-induced apoptosis through decreasing the level of cleaved caspase-3. In contrast, p38 MAPK, but not JNK1/2, was involved in the protoapigenone-mediated S and G(2)/M arrest by modulating the levels of Cdk2 and p-Cdc25C (Ser216). Moreover, in vivo xenograft study showed that protoapigenone had a significant inhibition of prostate tumor growth without major side effects on the mice we tested. This inhibition was associated with induction of apoptosis and activation of p38 MAPK and JNK1/2 in protoapigenone-treated tumor tissues. In conclusion, our results demonstrated protoapigenone suppressed prostate cancer cell growth through the activation of p38 MAPK and JNK1/2, with the potential to be developed as a chemotherapeutic agent for prostate cancer.     

Antitumor activity and molecular effects of the novel heat shock protein 90 inhibitor, IPI-504, in pancreatic cancer

Targeting Hsp90 is an attractive strategy for anticancer therapy because the diversity and relevance of biological processes are regulated by these proteins in most cancers. However, the role and mode of action of Hsp90 inhibitors in pancreatic cancer has not been studied. This study aimed to assess the antitumor activity of the Hsp90 inhibitor, IPI-504, in pancreatic cancer and to determine the biological effects of the agent. In vitro, we show that pharmacologic inhibition of Hsp90 by IPI-504 exerts antiproliferative effects in a panel of pancreatic cancer cells in a dose- and time-dependent manner. In pancreatic cancer xenografts obtained directly from patients with pancreas cancer, the agent resulted in a marked suppression of tumor growth. Although known Hsp90 client proteins were significantly modulated in IPI-504-treated cell line, no consistent alteration of these proteins was observed in vivo other than induction of Hsp70 expression in the treated xenografted tumors. Using a proteomic profiling analysis with isotope tags for relative and absolute quantitation labeling technique, we have identified 20 down-regulated proteins and 42 up-regulated proteins on IPI-504 treatment.tumor growth Identical changes were observed in the expression of the genes coding for these proteins in a subset of proteins including HSPA1B, LGALS3, CALM1, FAM84B, FDPS, GOLPH2, HBA1, HIST1H1C, HLA-B, and MARCKS. The majority of these proteins belong to the functional class of intracellular signal transduction, immune response, cell growth and maintenance, transport, and metabolism. In summary, we show that IPI-504 has potent antitumor activity in pancreatic cancer and identify potential pharmacologic targets using a proteomics and gene expression profiling.     

Enigmol: a novel sphingolipid analogue with anticancer activity against cancer cell lines and in vivo models for intestinal and prostate cancer

Sphingoid bases are cytotoxic for many cancer cell lines and are thought to contribute to suppression of intestinal tumorigenesis in vivo by ingested sphingolipids. This study explored the behavior of a sphingoid base analogue, (2S,3S,5S)-2-amino-3,5-dihydroxyoctadecane (Enigmol), that cannot be phosphorylated by sphingosine kinases and is slowly N-acylated and therefore is more persistent than natural sphingoid bases. Enigmol had potential anticancer activity in a National Cancer Institute (NCI-60) cell line screen and was confirmed to be more cytotoxic and persistent than naturally occurring sphingoid bases using HT29 cells, a colon cancer cell line. Although the molecular targets of sphingoid bases are not well delineated, Enigmol shared one of the mechanisms that has been found for naturally occurring sphingoid bases: normalization of the aberrant accumulation of β-catenin in the nucleus and cytoplasm of colon cancer cells due to defect(s) in the adenomatous polyposis coli (APC)/β-catenin regulatory system. Enigmol also had antitumor efficacy when administered orally to Min mice, a mouse model with a truncated APC gene product (C57Bl/6J(Min/+) mice), decreasing the number of intestinal tumors by half at 0.025% of the diet (w/w), with no evidence of host toxicity until higher dosages. Enigmol was also tested against the prostate cancer cell lines DU145 and PC-3 in nude mouse xenografts and suppressed tumor growth in both. Thus, Enigmol represents a novel category of sphingoid base analogue that is orally bioavailable and has the potential to be effective against multiple types of cancer.     

Antitumor activity of CTFB, a novel anticancer agent, is associated with the down-regulation of nuclear factor-kappaB expression and proteasome activation in head and neck squamous carcinoma cell lines

This study aimed to characterize the antitumor activity of 5-Chloro-N-[2-[2-(4-chloro-phenyl)-3-methyl-butoxy]-5-trifluoromethyl-phenyl]-2-hydroxy-benzamide (CTFB), a novel anticancer agent, in head and neck cancer cell lines, FaDu, SCC-25 and cisplatin-resistant CAL-27. CTFB was generated as a result of an extensive medicinal chemistry effort on a lead compound series discovered in a high-throughput screen for inducers of apoptosis. All cell lines showed significant growth delay in response to CTFB treatment at a concentration of 1 micromol/L with 17.16 +/- 2.08%, 10.92 +/- 1.22%, and 27.03 +/- 1.86% of cells surviving at 120 h in FaDu, CAL-27, and SCC-25, respectively. To define proteins involved in the mechanism of action of CTFB, we determined differences in the proteome profile of cell lines before and after treatment with CTFB using two-dimensional difference gel electrophoresis followed by computational image analysis and mass spectrometry. Eight proteins were found to be regulated by CTFB in all cell lines. All these proteins are involved in cytoskeleton formation and function and/or in cell cycle regulation. We showed that CTFB-induced cell growth delay was accompanied by cell cycle arrest at the G(0)-G(1) phase that was associated with the up-regulation of p21/WAF1 and p27/Kip1 expression and the down-regulation of cyclin D1. Furthermore, we showed that activity of CTFB depended on the down-regulation of nuclear factor-kappaB (NF-kappaB) and NF-kappaB p65 phosphorylated at Ser(536). The level of proteasome activity correlated with the response to CTFB treatment, and the down-regulation of NF-kappaB is accompanied by enhanced proteasome activity in all investigated head and neck cancer cell lines. In this report, we show that CTFB reveals multiple effects that lead to delayed cell growth. Our data suggest that this compound should be studied further in the treatment of head and neck cancer.