Donald M. Miller

Antiproliferative Activity of G-rich Oligonucleotides Correlates with Protein Binding

Oligonucleotides have been extensively studied as antisense or antigene agents that can potentially modulate the expression of specific genes. These strategies rely on sequence-specific hybridization of the oligonucleotide to mRNA or genomic DNA. Recently, it has become clear that oligonucleotides often have biological activities that cannot be attributed to their sequence-specific interactions with nucleic acids. Here we describe a series of guanosine-rich phosphodiester oligodeoxynucleotides that strongly inhibit proliferation in a number of human tumor cell lines. The presence of G-quartets in the active oligonucleotides is demonstrated using an UV melting technique. We show that G-rich oligonucleotides bind to a specific cellular protein and that the biological activity of the oligonucleotides correlates with binding to this protein. The G-rich oligonucleotide-binding protein was detected in both nuclear and cytoplasmic extracts and in proteins derived from the plasma membrane of cells. We present strong evidence that this protein is nucleolin, a multifunctional phosphoprotein whose levels are related to the rate of cell proliferation. Our results indicate that binding of G-rich oligonucleotides to nucleolin may be responsible for their non-sequence-specific effects. Furthermore, these oligonucleotides represent a new class of potentially therapeutic agents with a novel mechanism of action.

Structural Analysis of α-Enolase MAPPING THE FUNCTIONAL DOMAINS INVOLVED IN DOWN-REGULATION OF THE c-myc PROTOONCOGENE

Myc-binding protein-1 (MBP-1) is a 37-kDa protein with sequence homology to the 3′ portion of the α-enolase gene. α-Enolase is a 48-kDa protein, which plays a critical role in the glycolytic pathway. MBP-1 binds to the c-myc P2 promoter and down-regulates c-myc expression. We have investigated the role of α-enolase in regulation of the c-mycprotooncogene. RNase protection assay shows that α-enolase is transcribed into a single RNA species in HeLa cells. A start codon, 400 base pairs downstream of the α-enolase ATG, corresponds to the MBP-1 ATG, suggesting that MBP-1 is an alternative translation initiation product of the α-enolase RNA. Domain mapping was performed using constructs containing truncations of the α-enolase gene. In vitro binding to the c-myc gene was abolished after deletion of the N-terminal portion of α-enolase. In order to determine the relationship between DNA binding activity and transcription inhibition, we performed co-transfection assays in HeLa cells. These studies confirmed that an N-terminal deletion of α-enolase is unable to down-regulate c-myc promoter activity. Our data suggest that α-enolase plays an important role in regulation of c-myc promoter activity in the form of an alternative translation product MBP-1, which is distinct from its role as a glycolytic enzyme.

c-myc amplification in ovarian cancer

The c-myc oncogene codes for a DNA binding protein that appears to play an important role in the regulation of cell growth. c-myc gene amplification has been documented to occur in both hematopoietic and solid neoplasms and often indicates more biologically aggressive tumors. Southern hybridization analysis was performed on high-molecular-weight DNA isolated from primary ovarian carcinomas. Major structural rearrangements of c-myc were not detected. Five of seventeen (29.4%) tumor samples demonstrated amplification of the myc oncogene. The 5 patients with ovarian carcinomas associated with c-myc amplification exhibited a median survival of 17 months. Of the 12 patients without evidence of tumor-associated c-myc amplification, 5 have exhibited disease-free survival for an average of 36.8 months and are currently alive. The remaining 7 patients, the majority of whom had advanced-stage, poorly differentiated lesions with a normal c-myc copy number, exhibited a median survival of 9 months. There was no apparent relationship between c-myc amplification, grade of tumor differentiation, and response to platinol-based chemotherapy. These data do not suggest a prognostic role for c-myc amplification in primary ovarian cancer. However, c-myc amplification is a common finding in advanced-stage ovarian cancer.

The G-C Specific DNA Binding Drug, Mithramycin, Selectively Inhibits Transcription of the C-MYC and C-HA-RAS Genes in Regenerating Liver

Expression of the c-myc and c-Haras protooncogenes is dramatically increased in regenerating rat liver as an early response to partial hepatectomy. Nuclear runon transcription studies confirm that the increased c-myc and c-Ha-ras mRNA levels in regenerating livers reflect transcriptional activation of these genes. Mithramycin, a G-C specific DNA binding drug, prevents the increased transcriptional activity of c-myc and c-Ha-ras genes after hepatectomy but does not alter the transcriptional activity of the beta-actin gene. Continuous exposure of rats to mithramycin after hepatectomy prevents the increase in both c-myc and c-Ha-ras expression and blocks the increased cellular proliferation characteristic of regeneration. The delayed increase in c-myc and c-Ha-ras gene expression is associated with a delay in cellular proliferation. The inhibition of c-myc and c-Ha-ras transcription by mithramycin, the delay in cellular proliferation, and the ability of mithramycin to prevent protein binding to the c-myc promoter, suggest that the increased expression of these genes is a necessary component of liver regeneration.

Structure and applications of intermolecular DNA triplexes.

Current DNA binding drugs are not sequence specific. Triplex-forming oligonucleotides will bind targeted duplex DNA sites in a sequence-specific manner. A new class of DNA binding molecules based on triple-helical DNA formation promises a sequence-specific method of targeting discrete regions of DNA. DNA modifying molecules linked to third strands have been shown to modify only regions of DNA to which they were targeted. Current research will increase the understanding of triplex DNA structure and will lead to improved DNA binding drugs.

Selective inhibition of c-myc expression by the ribonucleic acid synthesis inhibitor mithramycin☆

Expression of the c-myc proto-oncogene has been shown to correlate with the rate of cellular proliferation and malignant transformation in a number of cell types. JEG-3 choriocarcinoma cells demonstrate c-myc transcript levels that are greater than those of nonmalignant trophoblastic tissue at any stage of gestation. Southern blot analysis documents c-myc gene amplification in JEG-3 cells, with a gene copy number of approximately 20. The methylation pattern and genomic structure of the amplified c-myc oncogene in JEG-3 choriocarcinoma cells are identical to those of normal placenta. Treatment of JEG-3 cells with mithramycin, a ribonucleic acid synthesis inhibitor, results in a dramatic decrease in c-myc expression relative to that of the c-Ha-Ras gene. The apparent selectivity of mithramycin for c-myc expression represents the only example, to date, of the selective pharmacologic modulation of oncogene expression.

ROLE OF IMMUNE CELLS IN THE PAVLOVIAN CONDITIONING OF SPECIFIC RESISTANCE TO CANCER

It has been demonstrated that significant protection against YC8 lymphoma can be induced in mice preimmunized with normal DBA/2 spleen cells. The DBA/2 spleen cells used as alloantigens share minor histocompatibility determinants with the YC8 tumor. We have used this model to investigate the nature of the immunity conferred by treatment with the alloantigen and infer that the conditioned resistance observed was maintained by the same effector mechanism. The results demonstrated that repeated immunization of tumor bearing mice with the alloantigen had some beneficial effect as shown by the slower rate of growth of the tumor, and an increase in median survival time over controls. The observations showed however that once tumor was present in vivo, the use of potent tumor specific vaccine can help in increasing survival but can no longer produce high incidence of regressions and cures. Conditioning can potentiate the effects of this treatment by increasing survival and cure.

Mithramycin Selectively Inhibits the Transcriptional Activity of a Transfected Human C-myc Gene

The G-C specific DNA binding drug mithramycin selectively inhibits expression of the c-myc gene in a number of cell types. We have tested the ability of this agent to inhibit the expression of a transfected human c-myc gene in a murine fibroblast cell line. Expression of c-myc is inhibited in the first 24 hours of mithramycin exposure (in contrast to actin gene expression, which is unaffected). Nuclear runon transcription of c-myc by nuclei isolated from mithramycin treated cells is decreased, indicating inhibition of transcription initiation. However, treatment of isolated nuclei with mithramycin also results in decreased c-myc transcription. Thus, inhibition of c-myc expression by mithramycin in these cells appears to occur at the transcriptional level and is most likely mediated at both the transcription initiation and elongation level. This suggests that mithramycin selectively interacts with the G-C rich c-myc promoter, preventing formation of the c-myc transcription initiation complex.

Oncogenes, Malignant Transformation, and Modern Medicine

During the past decade there have been remarkable strides in the understanding of the basic mechanism of cancer. It is now clear that there is a set of genes, known as oncogenes, that can cause cells to become malignant if their expression is altered, either by mutation or overexpression. The products of these genes include growth factors, growth factor receptors, signal tranduction proteins, and DNA binding proteins. The normal cellular counterparts of these genes play very important roles in the regulation of growth and proliferation by normal cells. Another set of genes, anti-oncogenes, also play an important role in preventing abnormal cell proliferation. The remarkable explosion of understanding of the pathophysiology of malignancy has led to a common unifying concept of malignant transformation that applies to all tumors. It is likely that these new insights will lead to improved and more specific treatments for malignant disease in the next decade.

Review: Basic Mechanisms of Metastasis

ABSTRACT Metastatic disease is responsible for the majority of deaths caused by cancer. The process of metastasis is an orderly, stepwise process that results in the selection of cells that possess the capability to establish viable metastases. These cells must be locally invasive and be able to survive the physical traumas of dissemination and normal host defenses. Once metastatic cells have been arrested in a capillary bed, they must be able to invade the host organ parenchyma and survive in that milieu. Studies in a number of model systems have documented the phenotypic alterations in cells that have “metastatic potential.” These differences may stem from normal tumor cell heterogeneity and surprisingly reflect only minor differences in gene expression. The role of activated oncogenes in metastasis is unclear, but a number of laboratories have documented that transfection with activated Ha-Ras results in increased metastatic potential. An increased understanding of the genetic basis of metastatic potential may suggest new directions for intervening in this deadly process.