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Biomedical subjects

D Mercola

Publications and source records attributed to D Mercola.

At least 37 records · Page 2Linked to original sources
PubMed1999

Inhibition of the Jun kinase pathway blocks DNA repair, enhances p53-mediated apoptosis and promotes gene amplification.

We have previously shown, by expression of a nonphosphorylatable dominant inhibitor mutant of c-Jun [cJun(S63A,S73A)], that activation of the NH2-terminal Jun kinase/stress-activated protein kinase by genotoxic damage is required for DNA repair. Here, we examine the consequences of inhibition of DNA repair on p53-induced apoptosis in T98G cells, which are devoid of endogenous wild-type p53. Relative to parental or wild-type c-Jun-expressing control cells, mutant Jun-expressing T98G clones show similar growth rates and plating efficiencies. However, these cells are unable to repair DNA (PCR-stop assays) and exhibit up to an 80-fold increased methotrexate-induced colony formation due to amplification of the dihydrofolate reductase gene. Moreover, the mutant c-Jun clones exhibit increased apoptosis and elevated bax:bcl2 ratios on expression of wild-type p53. These results indicate that inhibition of DNA repair leads to accumulation of DNA damage in tumor cells with unstable genomes and this, in turn, enhances p53mediated apoptosis.

Apoptosis↗
PubMed1999

Interleukin 2 gene therapy of colorectal carcinoma with autologous irradiated tumor cells and genetically engineered fibroblasts: a Phase I study.

The purpose of this study was to determine the safety, toxicity, and antitumor immune response following S.C. immunizations with a mixture of irradiated, autologous tumor cells and autologous fibroblasts that were genetically modified to express the gene for interleukin 2 (IL-2) in patients with colorectal carcinoma. Ten patients were treated with a fixed dose of tumor cells (10(7)) and escalating doses of fibroblasts secreting IL-2 (per 24 h): 100 units (three patients), 200 units (three patients), 400 units (three patients), and 800 units (one patient). Pre- and posttreatment peripheral blood mononuclear cells were evaluated for evidence of antitumor immune responses. Fatigue and/or flu-like symptoms were experienced by seven patients and delayed-type hypersensitivity-like skin reactions were observed at the sites of the second or subsequent vaccinations in five patients. Low frequencies of tumor cytotoxic T-cell precursors (range, 1/190,000-1/1,320,000 peripheral blood mononuclear cells) were detected prior to therapy in four of seven patients. There was a 5-fold increase following treatment in the frequency of tumor cytotoxic T-cell precursors in two of six evaluable patients. Some patients with colorectal cancer have low frequencies of tumor cytotoxic T-cell precursors that may be increased by this well-tolerated form of IL-2 gene therapy, which warrants continued clinical evaluation.

Cancer Vaccines↗
PubMed1998

Molecular determinants of AHPN (CD437)-induced growth arrest and apoptosis in human lung cancer cell lines.

6-[3-(1-Adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN or CD437), originally identified as a retinoic acid receptor gamma-selective retinoid, was previously shown to induce growth inhibition and apoptosis in human breast cancer cells. In this study, we investigated the role of AHPN/CD437 and its mechanism of action in human lung cancer cell lines. Our results demonstrated that AHPN/CD437 effectively inhibited lung cancer cell growth by inducing G0/G1 arrest and apoptosis, a process that is accompanied by rapid induction of c-Jun, nur77, and p21(WAF1/CIP1). In addition, we found that expression of p53 and Bcl-2 was differentially regulated by AHPN/CD437 in different lung cancer cell lines and may play a role in regulating AHPN/CD437-induced apoptotic process. On constitutive expression of the c-JunAla(63,73) protein, a dominant-negative inhibitor of c-Jun, in A549 cells, nur77 expression and apoptosis induction by AHPN/CD437 were impaired, whereas p21(WAF1/CIP1) induction and G0/G1 arrest were not affected. Furthermore, overexpression of antisense nur77 RNA in A549 and H460 lung cancer cell lines largely inhibited AHPN/CD437-induced apoptosis. Thus, expression of c-Jun and nur77 plays a critical role in AHPN/CD437-induced apoptosis. Together, our results reveal a novel pathway for retinoid-induced apoptosis and suggest that AHPN/CD437 or analogs may have a better therapeutic efficacy against lung cancer.

Apoptosis↗
PubMed1997

The JUN kinase/stress-activated protein kinase pathway is required for epidermal growth factor stimulation of growth of human A549 lung carcinoma cells.

Epidermal growth factor (EGF) plays a major role in non-small cell lung cancer cell autocrine growth and has been reported to activate the JUN kinase/stress-activated protein kinase (JNK/SAPK) pathway in model cells. Activation of JNK/SAPK leads to the phosphorylation of c-JUN protooncogene on serines 63 and 73. This mechanism is required for and cooperates in the transformation of rat embryo fibroblasts by Ha-RAS. However, the function of JNK/SAPK in human tumor growth is unknown. We have tested several lung carcinoma cell lines. All exhibited UV-C-inducible JNK/SAPK activity; two exhibited constitutive activity in low serum, and two (M103 and A549) exhibited EGF-inducible JNK/SAPK activity. In A549 cells, EGF induced a rapid and prolonged (up to 24 h) activation of the JNK/SAPK pathway that correlated with a 150-190% growth stimulation. Stably transfected clones of A549 cells expressing c-JUN(S63A,S73A), a transdominant inhibitor of c-JUN, completely blocked the EGF-stimulated proliferation effect but did not alter the basal proliferation rate. Consistent with these results JNK antisense oligonucleotides targeted to JNK1 and JNK2 entirely eliminated the EGF-stimulated JNK/SAPK activity and blocked EGF-stimulated growth but not basal growth. In contrast, specific inhibition of the RAF/ERK pathway by PD98059 (MEK1 inhibitor) completely blocked ERK activation by EGF and basal cell growth but not EGF-stimulated growth, thereby dissociating the growth-promoting roles of each pathway. Our observations indicate, for the first time, that JNK/SAPK may be a preferential effector pathway for the growth properties of EGF in A549 cells.

Animals↗
PubMed1997

Reciprocal modulation between Sp1 and Egr-1.

Many ubiquitously expressed genes, including oncogenes, lack a proximal TATA or CAAT box but have a region of G + C-rich sequences that appears to replace the usual promoter initiation site. The zinc-finger protein Sp1 is one of the prevalent activators of these genes. The Egr-1 zinc-finger protein has a similar binding site and if the two sites occur in the same region, a variety of activation or inhibitory responses may be obtained. We show that competition between the two factors for overlapping sites on growth-promoting genes could explain why the overexpression of Egr-1 suppresses transformed growth in a number of cell types [Huang et al. (1995): Cancer Res 55:5054-5062; Huang et al. (1997): Int J Cancer]. We demonstrate here that Egr-1 and Sp1 can bind to the same G + C-rich sites and that Egr-1 can displace Sp1 and hence inhibit its activity. We measured the responses of synthetic consensus binding sites and natural promoter sequences linked to a reporter gene and showed that Egr-1 inhibited the activation of transcription by Sp1 on overlapping Sp1/Egr-1 sites. In contrast, Sp1 activity could be augmented by Egr-1 at nonoverlapping sites in the Egr-1 gene promoter, in transient reporter gene studies in Drosophila SL2 cells. In addition, over-expression of exogenous Sp1 in mammalian cells, also leads to increased Egr-1 protein expression, which further inhibits Sp1 transactivation of numerous genes. Therefore, we can account for some of the complex responses of G + C-rich enhancer/promoters by a form of "facilitated inhibition" of Sp1 by Egr-1 at overlapping sites.

3T3 Cells↗
PubMed1997

Decreased Egr-1 expression in human, mouse and rat mammary cells and tissues correlates with tumor formation.

We have examined several types of tumor cell lines and shown that they invariably expressed little or no Egr-1, in contrast to their normal counterparts. We have previously shown that the expression of exogenous Egr-1 in human breast and other tumor cells markedly reduces transformed growth and tumorigenicity. We therefore hypothesized that the loss of Egr-1 expression plays a role in transformation. All human and mouse breast cancer cell lines and tumors examined had reduced Egr-1 expression compared with their normal counterparts. Reduced Egr-1 expression was also observed in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumors, and this level increased to normal levels in tumors that regressed after tamoxifen treatment. We concluded, therefore, that loss of Egr-1 expression may play a role in the deregulation of normal growth in the tumorigenic process and that Egr-1 acts as a tumor suppressor gene.

Animals↗
PubMed1997

The Jun kinase/stress-activated protein kinase pathway functions to regulate DNA repair and inhibition of the pathway sensitizes tumor cells to cisplatin.

We have studied the role of Jun/stress-activated protein kinase (JNK/SAPK) pathway in DNA repair and cisplatin resistance in T98G glioblastoma cells. JUN/SAPK is activated by DNA damage and phosphorylates serines 63 and 73 in the N-terminal domain of c-Jun, which is known to increase its transactivation properties. We show that treatment of T98G glioblastoma cells with cisplatin but not the transplatin isomer activates JNK/SAPK about 10-fold. T98G cells, which are highly resistent to cisplatin (IC50 = 140 +/- 13 microM), modified to express a nonphosphorylatable dominant negative c-Jun (termed dnJun) exhibit decreased viability following treatment with cisplatin, but not transplatin, in proportion (rPearson = 0.98) to the level of dnJun expressed leading to a 7-fold decreased IC50. Similar effects are observed in U87 cells, PC-3 cells, and MCF-7 cells, as well as in T98G cells modified to express TAM-67, a known inhibitor of c-Jun function. In contrast, no sensitization effect was observed in cells modified to express wild-type c-Jun. Furthermore, through quantitative polymerase chain reaction-stop assays, we show that dnJun expressing cells were inhibited in repair of cisplatin adducts (p = 0.55), whereas repair is readily detectable (p = 0.003) in parental cells. These observations indicate that the JNK/SAPK pathway is activated by cisplatin-induced DNA damage and that this response is required for DNA repair and viability following cisplatin treatment. Regulation of DNA repair following genotoxic stress may be a normal physiological role of the JNK/SAPK pathway.

Antineoplastic Agents↗
PubMed1996

Expression of Egr-1 correlates with the transformed phenotype and the type of viral latency in EBV genome positive lymphoid cell lines.

In this paper we have investigated the role of Egr-1 in B cell growth regulation by examining the gene expression in a panel of B cell lines, including both EBV genome negative and EBV carrying cell lines. Egr-1 expression correlates with the cellular phenotype and the specific pattern of viral latency established within the individual cell lines. Thus, constitutive activation of Egr-1 gene is invariably associated with unrestricted expression of viral latent genes in all group III EBV genome carrying cell lines. In contrast, Egr-1 expression is abrogated in group I Burkitt tumor cells, irrespective of the EBV genome carrying status. Activated viral gene expression associated with phenotypic conversion of group I cell lines in to group II or III restores the Egr-1 gene expression. Several forms of EGR-1 protein are found within the different groups of cell lines, and the binding activity to DNA consensus sequences was investigated. Finally, time course analysis of Egr-1 expression during the early steps of EBV infection in vitro demonstrated that Egr-1 is upregulated within minutes from the initial interaction with the B lymphocyte.

B-Lymphocytes↗
PubMed1996

Transcription factor EGR-1 suppresses the growth and transformation of human HT-1080 fibrosarcoma cells by induction of transforming growth factor beta 1.

The early growth response 1 (EGR-1) gene product is a transcription factor with role in differentiation and growth. We have previously shown that expression of exogenous EGR-1 in various human tumor cells unexpectedly and markedly reduces growth and tumorigenicity and, conversely, that suppression of endogenous Egr-1 expression by antisense RNA eliminates protein expression, enhances growth, and promotes phenotypic transformation. However, the mechanism of these effects remained unknown. The promoter of human transforming growth factor beta 1 (TGF-beta 1) contains two GC-rich EGR-1 binding sites. We show that expression of EGR-1 in human HT-1080 fibrosarcoma cells uses increased secretion of biologically active TGF-beta 1 in direct proportion (rPearson = 0.96) to the amount of EGR-1 expressed and addition of recombinant human TGF-beta 1 is strongly growth-suppressive for these cells. Addition of monoclonal anti-TGF-beta 1 antibodies to EGR-1-expressing HT-1080 cells completely reverses the growth inhibitory effects of EGR-1. Reporter constructs bearing the EGR-1 binding segment of the TGF-beta 1 promoter was activated 4- to 6-fold relative to a control reporter in either HT-1080 cells that stably expressed or parental cells cotransfected with an EGR-1 expression vector. Expression of delta EGR-1, a mutant that cannot interact with the corepressors, nerve growth factor-activated factor binding proteins NAB1 and NAB2, due to deletion of the repressor domain, exhibited enhanced transactivation of 2- to 3.5-fold over that of wild-type EGR-1 showing that the reporter construct reflected the appropriate in vivo regulatory context. The EGR-1-stimulated transactivation was inhibited by expression of the Wilms tumor suppressor, a known specific DNA-binding competitor. These results indicate that EGR-1 suppresses growth of human HT-1080 fibrosarcoma cells by induction of TGF-beta 1.

Binding Sites↗
PubMed1996

Growth factor PDGF-B/v-sis confers a tumorigenic phenotype to human tumor cells bearing PDGF receptors but not to cells devoid of receptors: evidence for an autocrine, but not a paracrine, mechanism.

Numerous established human tumor lines co-express platelet-derived growth factor (PDGF) and cognate receptors, suggesting that an autocrine and/or paracrine growth mechanism may be a causal or contributing mechanism to their transformed phenotype. Indeed, it is known that a PDGF-autocrine system is functional in several established tumor lines, especially in human gliomas, and a model for a functional paracrine mechanism has been established in a human melanoma line. However, at least 168 human cell lines representing 26 different human tumor types have been reported to continuously express PDGF-A and/or -B chains, and 55 of these also express PDGF receptors. For the majority of these cases, the significance of co-expression and the relative roles of autocrine and paracrine mechanisms in transformation remains unclear. Here, we show that human glioblastoma T98G cells co-express PDGF-B/c-sis and moderate levels of the cognate beta-type PDGF receptor (PR-beta) but are not tumorigenic in athymic mice. In contrast, human breast carcinoma MCF-7 cells do not express PR-beta and are tumorigenic. Clonal lines of each cell type with greatly increased secretion of p16w(T98Gsis and MCF-7sis cells) were characterized. T98Gsis cells are 85% tumorigenic and occasionally develop pulmonary metastases, showing that endogenous PR-beta can mediate complete transformation upon sufficient stimulation. In contrast, MCF-7sis cells exhibit some growth slowing in vitro and an exactly proportional decrease in tumor growth rate. We conclude that a PDGF-autocrine, and not a paracrine, mechanism best accounts for the acquired tumorigenicity of T98Gsis cells, thereby emphasizing the potential significance of expression of even moderate levels of PR-beta by human tumor cells.

Animals↗
PubMed1996

Eradication of established intracranial rat gliomas by transforming growth factor beta antisense gene therapy.

Like human gliomas, the rat 9L gliosarcoma secretes the immunosuppressive transforming growth factor beta (TGF-beta). Using the 9L model, we tested our hypothesis that genetic modification of glioma cells to block TGF-beta expression may enhance their immunogenicity and make them more suitable for active tumor immunotherapy. Subcutaneous immunizations of tumor-bearing animals with 9L cells genetically modified to inhibit TGF-beta expression with an antisense plasmid vector resulted in a significantly higher number of animals surviving for 12 weeks (11/11, 100%) compared to immunizations with control vector-modified 9L cells (2/15, 13%) or 9L cells transduced with an interleukin 2 retroviral vector (3/10, 30%) (P < 0.001 for both comparisons). Histologic evaluation of implantation sites 12 weeks after treatment revealed no evidence of residual tumor. In vitro tumor cytotoxicity assays with lymph node effector cells revealed a 3- to 4-fold increase in lytic activity for the animals immunized with TGF-beta antisense-modified tumor cells compared to immunizations with control vector or interleukin 2 gene-modified tumor cells. These results indicate that inhibition of TGF-beta expression significantly enhances tumor-cell immunogenicity and supports future clinical evaluation of TGF-beta antisense gene therapy for TGF-beta-expressing tumors.

Animals↗
PubMed1996

Platelet-derived growth factor-B/v-sis confers a tumorigenic and metastatic phenotype to human T98G glioblastoma cells.

Autocrine stimulation by platelet-derived growth factor-B (PDGF-B)-like factors has been widely implicated as an important mechanism in the cause and/or maintenance of a variety of human tumors. However, normal human cells appear to be resistant to transformation by PDGF-B-like molecules, and a direct demonstration of the tumor-promoting or tumor-maintaining property of a PDGF-B autocrine system is lacking. T98G human glioblastoma cells are nontumorigenic in athymic mice. We show that these cells express predominantly PDGF-beta type receptors and continuously secrete small amount of PDGF-B/c-sis. Addition of suramin or specific anti-PDGF-B/v-sis antibody inhibits proliferation in culture. Conversely, multiple clonal lines that stably overexpress PDGF-B/v-sis (T98Gsis cells) exhibit a striking 200-250% increased proliferation rate and an enhanced colony-forming frequency in soft agar. Clonal lines with stable expression of PDGF-B/v-sis (T98Gsis cells) reliably (80%) develop tumors in 4-6 weeks, whereas the empty-vector control cells are nontumorigenic. Moreover, in some cases, T98Gsis cells disseminate to form bilateral and multifocal pulmonary metastases. The results show that T98G cells contain functional PDGF receptors that, upon sufficient stimulation, can cause greatly increased mitogenic response, which may account for the development of the malignant phenotype. Metastatic tumor formation in athymic mice by PDGF stimulation has not been reported previously. The mechanism may depend on preexisting changes such as the lost p53 function of these cells. T98Gsis cells provide a model of growth factor-dependent tumorigenesis and metastases, which may be helpful in elucidating these relationships.

Animals↗
PubMed1996

EGR-1, the reluctant suppression factor: EGR-1 is known to function in the regulation of growth, differentiation, and also has significant tumor suppressor activity and a mechanism involving the induction of TGF-beta1 is postulated to account for this suppressor activity.

The transcription factor EGR-1 is a potential regulator of over 30 genes and plays a role in growth, development, and differentiation and, in addition, has significant transformation suppression activity. The regulatory properties are reviewed and a hypothesis for the transformation suppression activity is proposed. EGR-1 contains three "zinc-finger" motifs in the C-terminal portion of the molecule that constitute the DNA-binding domain and interact with the promoters by virtue of two classes of GC-rich elements: single GC-elements (GCEs) with the consensus 5'-T-G-C-G-T/g-G/A-G-G-C/a/t-G-G/T-3' and overlapping sites consisting of an Sp-1 binding site and the GCE consensus or close homolog of these sequences. The Wilm's tumor suppressor gene product WT1 interacts with the same GCE and, owing in part to four alternate splice products, interacts with a broader range of GC-rich elements with the consensus 5'-GNGNGGGNG-3' and 5'-TCCTCCTCCTCCTC-3'. WT1 commonly but not invariably acts as repressor of transcription, whereas EGR-1, in the absence of overlapping Sp-1 binding sequences, is often an activator. The well-known rapid response of the EGR-1 gene following mitogenic stimulation together with the occurrence of GCEs in the promoters of many growth factors and protooncogenes suggests a role of EGR-1 in growth. Moreover, EGR-1 is constitutively expressed in several viral-transformed systems. On the other hand, studies of model and human tumor lines reveal that EGR-1 has significant growth and transformation suppression roles. Recent studies show that this effect can be accounted for by the ability of EGR-1 to induce the expression and secretion of TGF-beta1, a potent growth suppressor of many cell types, by binding to a single GCE of the TGF-beta1 promoter. Although the effects of EGR- at overlapping Sp1/EGR-1 DNA binding sites are not predictable, known cases fall into two loose groups. Sp1 is usually activating and increasing concentrations of EGR-1 lead to displacement that results in either inhibition of transactivation or EGR-1-dependent transactivation. Moreover, recent studies suggest that displaced Sp1 binds to and activates the endogenous Egr-1 gene, thereby leading to "facilitated inhibition" of Sp1 function by the resulting increased EGR-1. This effect may augment the growth suppressive function of EGR-1 based on induction of TGF-beta1.

Animals↗
PubMed1995

Egr-1 negatively regulates human tumor cell growth via the DNA-binding domain.

Human HT1080 fibrosarcoma cells, subclone H4, express little or no Egr-1 (Zif/268, Krox 24), an early growth response gene encoding a transcription factor. Phorbol ester (but not serum) treatment only can elicit a small increase in Egr-1 expression in H4, in contrast to the normally rapid, high transient expression of Egr-1 observed after the addition of a wide range of stimulating agents to normal or immortalized cell lines. Because several human tumor cell lines express little Egr-1, we tested the hypothesis that this loss was causal to transformation. We report here that the expression of exogenous mouse Egr-1 in H4 cells inhibits transformed growth in a dose-dependent manner and significantly suppresses tumorigenicity in athymic mice. By overexpression of the fragment in Egr-1 that is responsible for its DNA-binding activity, the zinc-finger domain, we show that this domain has a similar activity. Moreover, the expression of antisense mRNA encoding the DNA-binding domain increases the transformed character of the H4 cells. One possible conclusion is that endogenous Egr-1-like genes perform growth-regulatory functions. Other human tumor lines are also growth suppressed by Egr-1 overexpression including ZR-75-1 breast carcinoma, U251 glioblastoma, and to a lesser extent, SAOS-2 osteosarcoma cells. These results are surprising in light of the "early growth response" character of Egr-1 but extend our earlier report of suppression of growth in v-sis-transformed NIH3T3 cells.

Animals↗
PubMed1995

Use of wild-type p53 to achieve complete treatment sensitization of tumor cells expressing endogenous mutant p53.

It is known that transfer of the wild-type p53 gene into p53-negative cells from transgenic mice increases their sensitivity to drug and radiation-induced apoptosis. However, unlike many human tumors, these transgenic cells do not express mutant p53, and it is not known from these earlier studies whether wild-type p53 dominates the effects of mutant p53 with respect to drug and radiation sensitivity. We addressed this question in glioblastoma, a disease characterized by an unusually high level of intrinsic resistance to therapy and poor prognosis: mean survival time from diagnosis is only about 1 yr. We introduced the gene for wild-type p53 into human T98G glioblastoma cells, which express endogenous mutant p53 but not wild-type p53. Stable transfectants that co-expressed mutant and wild-type p53 had enhanced sensitivity to cisplatin and gamma radiation, compared with parental cells, control vector-transduced cells, and transduced cells that had lost expression of wild-type p53. Transient wild-type p53 expression after high-efficiency gene transfer by a p53 adenovirus also sensitized the cells to cisplatin and correlated with the induction of apoptosis. The sensitization effect was also observed in p53 adenovirus-infected H23 small cell lung carcinoma cells, which express endogenous mutant p53. Therefore, wild-type p53 gene transfer has dominant effects over mutant p53 in sensitizing tumor cells to therapy, which supports the potential of p53 gene therapy to enhance the efficacy of traditional therapy.

Antineoplastic Agents↗
PubMed1995

Characterization of a new human glioblastoma cell line that expresses mutant p53 and lacks activation of the PDGF pathway.

We have established and characterized a new glioblastoma cell line, termed GT9, from a biopsy sample of a female adult patient with glioblastoma multiforme. The line has now undergone over 60 passages and has been successfully cultured after cryopreservation. Immunofluorescence analyses with a panel of monoclonal antibodies were positive for glial fibrillary acidic protein and vimentin, and negative for neurofilament, galactocerebroside, and fibronectin, a pattern typical of glial cells. Based on a tetraploid, the composite karyotype of GT9 cells included the loss of chromosome 10, gain of chromosome 7, and the presence of double minute chromosomes, three of the most common karyotypic abnormalities in glioblastoma. Sequence analysis of p53 cDNA revealed a homozygous double mutation at codon 249 (commonly mutated in aflatoxin-associated hepatocellular carcinoma) and codon 250. Moreover, there was a complete absence of wild-type p53. However, unlike the majority of human glioblastomas previously described, the expression of platelet-derived growth factor-B (PDGF-B), a potent mitogenic autocrine factor, was low in GT9 cells. The expression and phosphorylation of c-Jun and Jun-B, downstream mediators of the PDGF pathway, were also low. Thus, deregulation of the PDGF pathway does not appear to be involved in the pathogenesis of the GT9 glioblastoma. Conversely, Jun-D, a negative regulator of cell growth, was also low. In addition, Phosphorylated Egr-1, a recently reported suppressor of PDGF-B/v-sis-transformed cells, was also low, suggesting that the lack of activation of the PDGF pathway was not due to these suppressive mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗