Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation

Histone deacetylases (HDACs) catalyze the removal of acetyl groups on the amino-terminal lysine residues of core nucleosomal histones. This activity is associated generally with transcriptional repression. We have reported previously that inhibition of HDAC activity by hydroxamic acid-based hybrid polar compounds, such as suberoylanilide hydroxamic acid (SAHA), induces differentiation and/or apoptosis of transformed cells in vitro and inhibits tumor growth in vivo. SAHA is a potentially new therapeutic approach to cancer treatment and is in Phase I clinical trials. In several tumor cell lines examined, HDAC inhibitors alter the expression of less than 1% of expressed genes, including the cell cycle kinase inhibitor p21WAF1. In T24 bladder carcinoma cells, SAHA induces up to a 9-fold increase in p21WAF1 mRNA and protein, which is, at least in part, because of an increase in the rate of transcription of the gene. SAHA causes an accumulation of acetylated histones H3 and H4 in total cellular chromatin by 2 h, which is maintained through 24 h of culture. An increase in the accumulation of acetylated H3 and H4 was detected throughout the p21WAF1 promoter and the structural gene after culture with SAHA. The level of histone acetylation did not change in chromatin associated with the actin and p27 genes, and their mRNA expression was not altered during culture of T24 cells with SAHA. Thus, the present findings indicate that the induction of p21WAF1 by SAHA is regulated, at least in part, by the degree of acetylation of the gene-associated histones and that this induced increase in acetylation is gene selective.

Tissue-engineered cells producing complex recombinant proteins inhibit ovarian cancer in vivo

Techniques of tissue engineering and cell and molecular biology were used to create a biodegradable scaffold for transfected cells to produce complex proteins. Mullerian Inhibiting Substance (MIS) causes regression of Mullerian ducts in the mammalian embryo. MIS also causes regression in vitro of ovarian tumor cell lines and primary cells from ovarian carcinomas, which derive from Mullerian structures. In a strategy to circumvent the complicated purification protocols for MIS, Chinese hamster ovary cells transfected with the human MIS gene were seeded onto biodegradable polymers of polyglycolic acid fibers and secretion of MIS confirmed. The polymer-cell graft was implanted into the right ovarian pedicle of severe combined immunodeficient mice. Serum MIS in the mice rose to supraphysiologic levels over time. One week after implantation of the polymer-cell graft, IGROV-1 human tumors were implanted under the renal capsule of the left kidney. Growth of the IGROV-1 tumors was significantly inhibited in the animals with a polymer-cell graft of MIS-producing cells, compared with controls. This novel MIS delivery system could have broader applications for other inhibitory agents not amenable to efficient purification and provides in vivo evidence for a role of MIS in the treatment of ovarian cancer.

Myc represses the p21(WAF1/CIP1) promoter and interacts with Sp1/Sp3

The cyclin-dependent kinase inhibitor p21(WAF1/CIP1) inhibits proliferation both in vitro and in vivo, and overexpression of p21 in normal and tumor cell lines results in cell cycle arrest. In contrast, ectopic expression of Myc alleviates G1 cell cycle arrest. Recent studies showed that Myc can repress p21 transcription, thereby overriding a p21-mediated cell cycle checkpoint. We found that activation of a Myc-estrogen receptor fusion protein by 4-hydroxytamoxifen in mouse cells resulted in suppression of endogenous p21 transcription. This effect was observed in the absence of de novo protein synthesis and was independent of histone deacetylase activity. In transient transfection studies, Myc effectively repressed p21 promoter constructs containing only 119 bp of sequence upstream of the transcription start site. This region contains multiple Sp1-binding sites and a potential initiator element, but no canonical Myc DNA-binding sites. Deletion of the potential initiator element does not affect repression of the p21 promoter by c-Myc. Coimmunoprecipitation and glutathione S-transferase pull-down experiments demonstrate that c-Myc may form complexes with Sp1/Sp3. We found that the central region of c-Myc interacts with the zinc finger domain of Sp1. Because Sp1 is required for p21 transcription, it is possible that Myc may down-regulate p21 transcription, at least in part, by sequestering Sp1. Repression of the p21 promoter may contribute to the ability of c-Myc to promote cell proliferation.

A candidate live inactivatable attenuated vaccine for AIDS.

The recent discovery of long term AIDS nonprogressors who harbor nef-attenuated HIV suggests that a naturally occurring live vaccine for AIDS may already exist. Animal models have shown that a live vaccine for AIDS, attenuated in nef, is the best candidate vaccine. There are considerable risks, real and perceived, with the use of live HIV vaccines. We have introduced a conditional lethal genetic element into HIV-1 and simian immunodeficiency virus (SIV) molecular clones deleted in nef. The antiviral strategy we employed targets both virus replication and the survival of the infected cell. The suicide gene, herpes simplex virus thymidine kinase (tk), was expressed and maintained in HIV over long periods of time. Herpes simplex virus tk confers sensitivity to the antiviral activity of acyclic nucleosides such as ganciclovir (GCV). HIV-tk and SIV-tk replication were sensitive to GCV at subtoxic concentrations, and virus-infected cells were eliminated from tumor cell lines as well as primary cell cultures. We found the HIV-tk virus to be remarkably stable even after being cultured in media containing a low concentration of GCV and then challenged with the higher dose and that while GCV resistant escape mutants did arise, a significant fraction of the virus remained sensitive to GCV.

Suppression of tumorigenicity by the wild-type tuberous sclerosis 2 (Tsc2) gene and its C-terminal region.

The Tsc2 gene, which is mutationally inactivated in the germ line of some families with tuberous sclerosis, encodes a large, membrane-associated GTPase activating protein (GAP) designated tuberin. Studies of the Eker rat model of hereditary cancer strongly support the role of Tsc2 as a tumor suppressor gene. In this study, the biological activity of tuberin was assessed by expressing the wild-type Tsc2 gene in tumor cell lines lacking functional tuberin and also in rat fibroblasts with normal levels of endogenous tuberin. The colony forming efficiency of Eker rat-derived renal carcinoma cells was significantly reduced following reintroduction of wild-type Tsc2. Tumor cells expressing the transfected Tsc2 gene became more anchorage-dependent and lost their ability to form tumors in severe combined immunodeficient mice. At the cellular level, restoration of tuberin expression caused morphological changes characterized by enlargement of the cells and increased contact inhibition. As with the full-length Tsc2 gene, a clone encoding only the C terminus of tuberin (amino acids 1049-1809, including the GAP domain) was capable of reducing both colony formation and in vivo tumorigenicity when transfected into the Eker rat tumor cells. In normal Rat1 fibroblasts, conditional overexpression of tuberin also suppressed colony formation and cell growth in vitro. These results provide direct experimental evidence for the tumor suppressor function of Tsc2 and suggest that the tuberin C terminus plays an important role in this activity.

Normal human serum contains a natural IgM antibody cytotoxic for human neuroblastoma cells.

Neuroblastoma (NB) is characterized by the second highest spontaneous regression of any human malignant disorder, a phenomenon that remains to be elucidated. In this study, a survey of 94 normal human adult sera revealed a considerable natural humoral cytotoxicity against human NB cell lines in approximately one-third of the tested sera of both genders. Specific cell killing by these sera was in the range of 40% to 95%. Serum cytotoxicity was dependent on an intact classical pathway of complement. By several lines of evidence, IgM antibodies were identified as the cytotoxic factor in the sera. Further analyses revealed that a 260-kDa protein was recognized by natural IgM of cytotoxic sera in Western blots of NB cell extracts. The antigen was expressed on the surface of seven human NB cell lines but not on human melanoma or other control tumor cell lines derived from kidney, pancreas, colon, bone, skeletal muscle, lymphatic system, and bone marrow. Furthermore, no reactivity was observed with normal human fibroblasts, melanocytes, and epidermal keratinocytes. The antigen was expressed in vivo as detected by immunohistochemistry in both the tumor of a NB patient and NB tumors established in nude rats from human NB cell lines. Most interestingly, the IgM anti-NB antibody was absent from the sera of 11 human NB patients with active disease. The anti-NB IgM also could not be detected in tumor tissue obtained from a NB patient. Collectively, our data suggest the existence of a natural humoral immunological tumor defense mechanism, which could account for the in vivo phenomenon of spontaneous NB tumor regression.

Eradication of human hepatic and pulmonary melanoma metastases in SCID mice by antibody-interleukin 2 fusion proteins.

Antibody-cytokine fusion proteins combine the unique targeting ability of antibodies with the multifunctional activity of cytokines. Here, we demonstrate the therapeutic efficacy of such constructs for the treatment of hepatic and pulmonary metastases of different melanoma cell lines. Two antibody-interleukin 2 (IL-2) fusion proteins, ch225-IL2 and ch14.18-IL2, constructed by fusion of a synthetic sequence coding for human IL-2 to the carboxyl end of the Cgamma1 gene of the corresponding antibodies, were tested for their therapeutic efficacy against xenografted human melanoma in vivo. Tumor-specific fusion proteins completely inhibited the growth of hepatic and pulmonary metastases in C.B-17 scid/scid mice previously reconstituted with human lymphokine-activated killer cells, whereas treatment with combinations of the corresponding antibodies plus recombinant IL-2 only reduced the tumor load. Even when treatment with fusion proteins was delayed up to 8 days after inoculation of tumor cells, it still resulted in complete eradication of micrometastases that were established at that time point. Selection of tumor cell lines expressing or lacking the targeted antigen of the administered fusion protein proved the specificity of the observed antitumor effect. Biodistribution analysis demonstrated that the tumor-specific fusion protein accumulated not only in subcutaneous tumors but also in lungs and livers affected with micrometastases. Survival times of animals treated with the fusion protein were more than doubled as compared to those treated with the combination of the corresponding antibody plus IL-2. Our data demonstrate that an immunotherapeutic approach using cytokines targeted by antibodies to tumor sites has potent effects against disseminated human melanoma.

Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53.

The tumor suppressor gene product p53 plays an important role in the cellular response to DNA damage from exogenous chemical and physical mutagens. Therefore, we hypothesized that p53 performs a similar role in response to putative endogenous mutagens, such as nitric oxide (NO). We report here that exposure of human cells to NO generated from an NO donor or from overexpression of inducible nitric oxide synthase (NOS2) results in p53 protein accumulation. In addition, expression of wild-type (WT) p53 in a variety of human tumor cell lines, as well as murine fibroblasts, results in down-regulation of NOS2 expression through inhibition of the NOS2 promoter. These data are consistent with the hypothesis of a negative feedback loop in which endogenous NO-induced DNA damage results in WT p53 accumulation and provides a novel mechanism by which p53 safeguards against DNA damage through p53-mediated transrepression of NOS2 gene expression, thus reducing the potential for NO-induced DNA damage.

Cloning and characterization of a cellular apoptosis susceptibility gene, the human homologue to the yeast chromosome segregation gene CSE1.

We recently isolated human cDNA fragments that render MCF-7 breast cancer cells resistant to cell death caused by Pseudomonas exotoxin, Pseudomonas exotoxin-derived immunotoxins, diphtheria toxin, and tumor necrosis factor. We report here that one of these fragments is an antisense fragment of a gene homologous to the essential yeast chromosome segregation gene CSE1. Cloning and analysis of the full-length cDNA of the human CSE1 homologue, which we name CAS for cellular apoptosis susceptibility gene, reveals a protein coding region with similar length (971 amino acids for CAS, 960 amino acids for CSE1) and 59% overall protein homology to the yeast CSE1 protein. The conservation of this gene indicates it has an important function in human cells consistent with the essential role of CSE1 in yeast. CAS is highly expressed in human tumor cell lines and in human testis and fetal liver, tissues that contain actively dividing cells. Furthermore, CAS expression increases when resting human fibroblasts are induced to proliferate and decreases when they are growth-arrested. Thus, CAS appears to play an important role in both toxin and tumor necrosis factor-mediated cell death, as well as in cell proliferation.

[Leu5]enkephalin-encoding sequences are targets for a specific DNA-binding factor.

A DNA-binding factor with high affinity and specificity for the [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes has been characterized. The factor has the highest affinity for the [Leu5]-enkephalin-encoding sequence in the dynorphin B-encoding region of the prodynorphin gene, has relatively high affinity for other [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes, but has no apparent affinity for similar DNA sequences coding for [Met5]-enkephalin in the prodynorphin or proopiomelanocortin genes. The factor has been named [Leu5]enkephalin-encoding sequence DNA-binding factor (LEF). LEF has a nuclear localization and is composed of three subunits of about 60, 70, and 95 kDa, respectively. The highest levels were observed in rat testis, cerebellum, and spleen and were generally higher in late embryonal compared to newborn or adult animals. LEF activity was also recorded in human clonal tumor cell lines. LEF inhibited the transcription of reporter genes in artificial gene constructs where a [Leu5]enkephalin-encoding DNA fragment had been inserted between the transcription initiation site and the coding region of the reporter genes. These observations suggest that the [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes also have regulatory functions realized through interaction with a specific DNA-binding factor.

Elevated expression of H type GDP-L-fucose:beta-D-galactoside alpha-2-L-fucosyltransferase is associated with human colon adenocarcinoma progression.

GDP-L-fucose:beta-D-galactoside alpha-2-L-fucosyltransferase (EC 2.4.1.69) is a key enzyme in the biosynthesis of fucosylated type 1 and 2 lactoseries structures, such as Lewis b and the H type 2 and Lewis Y, respectively, that are accumulated in colon adenocarcinoma. Analysis of the mRNA transcript level for the human H gene-encoded beta-D-galactoside alpha-2-L-fucosyltransferase revealed 40- and 340-fold increases in the mRNA levels in all adenocarcinomas and tumor cell lines, respectively, compared to normal colon mucosa where a low level of mRNA transcript was detected. A variable increase in mRNA transcript levels was observed in 50% of adenomatous polyps. Nucleotide sequence analysis of the protein coding region of the cDNAs derived from normal colon, adenoma, and colon adenocarcinoma revealed 100% homology, suggesting that there are no tumor-associated allelic variations within the H beta-D-galactoside alpha-2-L-fucosyltransferase cDNA. These results suggest that beta-D-galactoside alpha-2-L-fucosyltransferase expression highly correlates with malignant progression of colon adenocarcinoma.

Expression and methylation pattern of TSLC1 cascade genes in lung carcinomas

TSLC1 (tumor suppressor in lung cancer-1, IGSF4) encodes a member of the immunoglobulin superfamily molecules, which is involved in cell-cell adhesion. TSLC1 is connected to the actin cytoskeleton by DAL-1 (differentially expressed in adenocarcinoma of the lung-1, EPB41L3) and it directly associates with MPP3, one of the human homologues of a Drosophila tumor suppressor gene, Discs large. Recent data suggest that aberrant promoter methylation is important for TSLC1 inactivation in lung carcinomas. However, little is known about the other two genes in this cascade, DAL-1 and MPP3. Thus, we investigated the expression and methylation patterns of these genes in lung cancer cell lines, primary lung carcinomas and nonmalignant lung tissue samples. By reverse transcription-polymerase chain reaction, loss of TSLC1 expression was observed in seven of 16 (44%) non-small-cell lung cancer (NSCLC) cell lines and in one of 11 (9%) small-cell lung cancer (SCLC) cell lines, while loss of DAL- 1 expression was seen in 14 of 16 (87%) NSCLC cell lines and in four of 11 (36%) SCLC cell lines. By contrast, MPP3 expression was found in all tumor cell lines analysed. Similar results were obtained by microarray analysis. TSLC1 methylation was seen in 13 of 39 (33%) NSC LC cell lines, in one of 11 (9%) SCLC cell lines and in 100 of 268 (37%) primary NSCLCs. DAL-1 methylation was observed in 17 of 39 (44%) NSCLC cell lines, in three of 11 (27%) SCLC cell lines and in 147 of 268 (55%) primary NSCLCs. In tumors of NSCLC patients with stage II-III disease, DAL-1 methylation was seen at a statistically significant higher frequency compared to tumors of patients with stage I disease. A significant correlation between loss of expression and methylation of the genes in lung cancer cell lines was found. Overall, 65% of primary NSCLCs had either TSLC1 or DAL-1 methylated. Methylation of one of these genes was detected in 59% of NSCLC cell lines; however, in SCLC cell lines, methylation was much less frequently observed. The majority of nonmalignant lung tissue samples was not TSLC1 and DAL-1 methylated. Re-expression of TSLC1 and DAL-1 was seen after treatment of lung cancer cell lines with 5-aza-2$-deoxy-cytidine. Our results suggest that methylation of TSLC1 and/or DAL-1, leading to loss of their expression, is an important event in the pathogenesis of NSCLC.

A Therapeutic Antibody for Cancer, Derived from Single Human B Cells.

Some patients with cancer never develop metastasis, and their host response might provide cues for innovative treatment strategies. We previously reported an association between autoantibodies against complement factor H (CFH) and early-stage lung cancer. CFH prevents complement-mediated cytotoxicity (CDC) by inhibiting formation of cell-lytic membrane attack complexes on self-surfaces. In an effort to translate these findings into a biologic therapy for cancer, we isolated and expressed DNA sequences encoding high-affinity human CFH antibodies directly from single, sorted B cells obtained from patients with the antibody. The co-crystal structure of a CFH antibody-target complex shows a conformational change in the target relative to the native structure. This recombinant CFH antibody causes complement activation and release of anaphylatoxins, promotes CDC of tumor cell lines, and inhibits tumor growth in vivo. The isolation of anti-tumor antibodies derived from single human B cells represents an alternative paradigm in antibody drug discovery.

Design and Synthesis of 4-N-Alkanoyl and 4-N-Alkyl Gemcitabine Analogues Suitable for Positron Emission Tomography

Gemcitabine is a highly potent chemotherapeutic nucleoside agent used in the treatment of several cancers and solid tumors. However, it is therapeutically limitated because of toxicity to normal cells and its rapid intracellular deamination by cytidine deaminase into the inactive uracil derivative. Modification at the 4-(N) position of gemcitabine's exocyclic amine to an -amide functionality is a well reported prodrug strategy which has been that confers a resistance to intracellular deamination while also altering pharmacokinetics of the parent drug. Coupling of gemcitabine to carboxylic acids with varying terminal moieties afforded the 4-N-alkanoylgemcitabines whereas reaction of 4-N-tosylgemcitabine with the corresponding alkyl amines gave the 4-N-alkylgemcitabines. The 4-N-alkanoyl and 4-N-alkyl gemcitabine analogues with a terminal hydroxyl group on the 4-N-alkanoyl or 4-N-alkyl chain were efficiently fluorinated either with diethylaminosulfur trifluoride or under conditions that are compatible with the synthetic protocols for 18F labeling, such as displacement of the corresponding mesylate with KF/Kryptofix 2.2.2. The 4-N-alkanoylgemcitabine analogues displayed potent cytostatic activities against murine and human tumor cell lines with 50% inhibitory concentration (IC50) values in the range of low nM, whereas cytotoxicity of the 4-N-alkylgemcitabine derivatives were in the low to modest µM range. The cytostatic activity of the 4-N-alkanoylgemcitabines was reduced by several orders of magnitude in the 2'-deoxycytidine kinase (dCK)-deficient CEM/dCK- cell line while the 4-N-alkylgemcitabines were only lowered by 2-5 times. None of the 4-N-modified gemcitabines were found to be substrates for cytosolic dCK, however all were found to inhibit DNA synthesis. As such, the 4-N-alkanoyl gemcitabine derivatives likely need to be converted to gemcitabine prior to achieving their significant cytostatic potential, whereas the 4-N-alkylgemcitabines reach their modest activity without "measurable" conversion to gemcitabine. Thus, the 4-N-alkylgemcitabines provide valuable insight on the metabolism of 4-N-modified gemcitabine prodrugs.

Avaliação da atividade antitumoral de amidas graxas análogas de canabinóides

A atividade antiproliferativa in vitro de uma série de amidas graxas sintéticas, em sete linhagens de células tumorais foi investigada. Baseado em GI50, TGI e LC50, os ensaios preliminares mostraram que a maior parte dos compostos mostrou atividade antiproliferativa moderada a boa contra as linhagem de células tumorais testadas, principalmente em células de glioma humano (U251) e câncer de ovário humano com fenótipo de resistentencia a múltiplos fármacos (NCI-ADR/RES). A amida (R,S)-3d, derivada do ácido ricinoleico, mostrou uma elevada seletividade com potência de inibição do crescimento e morte celular para a linhagem de células de glioma. Além disso, as amidas (S)-3c e (S)-3e, derivadas dos ácidos oleico e linoleico respectivamente, foram especificas para glioma e ovário com fenótipo de resistência a múltiplos fármacos com inibição potente do crescimento celular. Estes resultados aliados a um perfil de segurança relativo quando analisado o efeito sobre as linhagens celulares não–tumorais, apontam para que estes compostos sirvam como modelos para o desenvolvimento de candidatos a fármacos para o tratamento de câncer, incluindo cânceres com fenótipo de resistência a múltiplos fármacos.