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Biomedical subjects

C A Stein

Publications and source records attributed to C A Stein.

At least 37 records · Page 2Linked to original sources
PubMed2003

G3139 (oblimersen) may inhibit prostate cancer cell growth in a partially bis-CpG-dependent non-antisense manner.

G3139 is an 18-mer phosphorothioate oligodeoxyribonucleotide, which is targeted to the initiation codon region of the bcl-2mRNA. Although treatment of PC3 prostate cancer cells with G3139, which contains two CpG motifs, causes a dramatic decrease in bcl-2 protein expression after 3 days, it did not result in significant cellular apoptosis, as it does in many other cell lines. The absence of apoptosis was demonstrated by the absence of pro-caspase 3 cleavage products and of Annexin V cell surface expression. In addition, ATP production and the mitochondrial membrane potential DeltaPsim were preserved. Despite this, G3139 significantly inhibited the rate of cellular proliferation in complete media and blocked cloning in soft agar. G4232, a variant of G3139 that down-regulates bcl-2 expression to the same extent but has both CpG cytidines C5 methylated, was only minimally antiproliferative. A series of mismatched G3139-related oligomers were synthesized that could also substantially down-regulate bcl-2 protein expression, but only if the CpG motifs were preserved, demonstrating the presence of additional non-antisense mechanisms. G3139 caused production of reactive oxygen species in growth-arrested cells and oxidation of nuclear guanosine to 8-hydroxy-2'-deoxyguanosine, as determined by 1F7 monoclonal antibody staining. Bromodeoxyuridine incorporation studies demonstrated that G3139 induced a G1-S entry block and an intra-S-phase block in PC3 cells that persisted as long as 3 days. This finding coincides with the observation that expression of several proteins encoded by S-phase genes, including c-myb and poly(ADP-ribose) polymerase, were significantly reduced. These results illustrate the complexity of the mechanism of action of G3139 in PC3 cells.

Agar↗
PubMed2002

Antisense RNA down-regulation of bcl-xL Expression in prostate cancer cells leads to diminished rates of cellular proliferation and resistance to cytotoxic chemotherapeutic agents.

bcl-xL is a M(r) 26,000 bcl-2 homologue that is highly expressed in prostate cancer cells. In previous studies, the down-regulation of its expression by antisense oligonucleotides led to resistance. In this work, the 445-bp 5' terminus of the bcl-xL cDNA was cloned in the antisense orientation and stably transfected into DU145 and LNCaP prostate cancer cells. In the DU145 (and to a lesser extent the LNCaP) transfectants, phenotypic changes (versus mock-transfected cells) included an increase in doubling time (from 36 to 175 h) in the clone in which bcl-xL protein expression was 25% of control. The transfectants did not demonstrate characteristic apoptotic changes, as demonstrated by 4',6-diamidino-2-phenylindole staining, lack of either DNA laddering, caspase-3 activation, or poly(ADP)ribose and lamin cleavage, and the absence of a significant sub-G(0) population. Cell cycle analysis demonstrated an increase in a tetraploid population (from 28% to 66%), as well as the appearance of a hypertetraploid population. Levels of cIAP-1 protein were almost undetectable in the mock cells but increased at least 25-fold in the DU145 transfectants. The down-regulation of bcl-xL in both DU145 (and to a much lesser extent in LNCaP) cells led to their resistance to cytotoxic agents, including docetaxel, mitoxantrone, etoposide, vinblastine, and carboplatin. Reversion of bcl-xL expression in stable DU145 transfectants to nearly the levels found in the mock-transfected cells was accomplished by retroviral infection of the cells with a bcl-xL sense cDNA under control of a prolific promoter. This led to a dramatic increase in the growth rate and in BrdUrd incorporation, as well as a sharp decrease in the expression of cIAP-1 protein. Overall, these findings highlight the adaptability of prostate cancer cells to loss of bcl-xL and suggest that in addition to its prosurvival role, bcl-xL protein may also be involved in the regulation of the rate of cellular proliferation.

Antineoplastic Agents↗
PubMed2002

Potential roles of antisense oligonucleotides in cancer therapy. The example of Bcl-2 antisense oligonucleotides.

Antisense oligonucleotides have been widely used to specifically and selectively downregulate gene expression at the messenger RNA level. Even though oligonucleotides are commonly used in laboratories and clinical trials, they can induce non-specific effects that can lead to misinterpretation of experimentally-derived results. This review summarizes precautions one should take when using oligonucleotides. In addition, the role of one oligonucleotide, G3139, which is targeted to the coding region of bcl-2 messenger RNA, in inhibiting tumor progression in vitro and in clinical trials, is described.

Animals↗
PubMed2002

Antisense therapy: recent advances and relevance to prostate cancer.

Currently employed treatment options for patients with advanced and metastatic cancer such as surgery, radiation, hormone therapy, and chemotherapy are limited. In particular, the well known limitations of chemotherapy are at least in part due to a lack of specificity. The activation of dominant oncogenes and inactivation of tumor suppressor genes may represent novel targets for cancer therapy. Antisense therapy has been widely used to specifically and selectively inhibit the expression of selected genes at the messenger RNA level. Combinations of antisense oligonucleotides with chemotherapeutic agents may offer important advantages in cancer treatment. Several antisense drugs, especially oblimersen (G3139), have shown interesting results in experiments in animals, and have entered clinical trials. However, control oligonucleotides must be carefully chosen to separate antisense effects from the many potential nonspecific effects of oligonucleotides. This review summarizes the advantages and limitations of antisense therapy and its use in the treatment of androgen-independent prostate cancer.

Apoptosis↗
PubMed2001

A combinatorial approach for selectively inducing programmed cell death in human pancreatic cancer cells.

Pancreatic cancer is an extremely aggressive neoplasm whose incidence equals its death rate. Despite intensive analysis, the genetic changes that mediate pancreatic cancer development and effective therapies for diminishing the morbidity associated with this disease remain unresolved. Through subtraction hybridization, we have identified a gene associated with induction of irreversible growth arrest, cancer reversion, and terminal differentiation in human melanoma cells, melanoma differentiation associated gene-7 (mda-7). Ectopic expression of mda-7 when using a recombinant adenovirus, Ad.mda-7, results in growth suppression and apoptosis in a broad spectrum of human cancers with diverse genetic defects, without exerting deleterious effects in normal human epithelial or fibroblast cells. Despite the apparently ubiquitous antitumor effects of mda-7, pancreatic carcinoma cells are remarkably refractory to Ad.mda-7 induced growth suppression and apoptosis. In contrast, the combination of Ad.mda-7 with antisense phosphorothioate oligonucleotides, which target the K-ras oncogene (a gene that is mutated in 85 to 95% of pancreatic carcinomas), induces a dramatic suppression in growth and a decrease in cell viability by induction of apoptosis. In mutant K-ras pancreatic carcinoma cells, programmed cell death correlates with expression and an increase, respectively, in MDA-7 and BAX proteins and increases in the ratio of BAX to BCL-2 proteins. Moreover, transfection of mutant K-ras pancreatic carcinoma cells with an antisense K-ras expression vector and infection with Ad.mda-7 inhibits colony formation in vitro and tumorigenesis in vivo in nude mice. These intriguing observations demonstrate that a combinatorial approach, consisting of a cancer-specific apoptosis-inducing gene and an oncogene inactivation strategy, may provide the foundation for developing an effective therapy for pancreatic cancer.

Adenoviridae↗
PubMed2001

The mononucleotide-dependent, nonantisense mechanism of action of phosphodiester and phosphorothioate oligonucleotides depends upon the activity of an ecto-5'-nucleotidase.

Many reports indicate different nonantisense yet sequence-specific effects of antisense phosphorothioate oligonucleotides. Products of enzymatic degradation of the oligonucleotides can also influence cell proliferation. The cytotoxic effects of deoxyribonucleoside-5'-phosphates (dNMPs) and their 5'-phosphorothioate analogs, deoxyribonucleoside-5'-monophosphorothioates (dNMPSs) on 4 human cell types (HeLa, HL-60, K-562, and endothelial cells) were examined, and the effects were correlated with the catabolism of these compounds. The results indicate that differences in cytotoxicity of dNMPs or dNMPSs in these cells depend upon different activity of an ecto-5'-nucleotidase. It has also been found that dNMPSs stimulate proliferation of human umbilical vein endothelial cells and HL-60 cells in a concentration-dependent manner. This stimulation might be caused by the binding of deoxynucleoside-5'-phosphorothioates to as-yet unidentified nucleotide receptor(s) at the cell surface. (Blood. 2001;98:995-1002)

5'-Nucleotidase↗
PubMed2001

Inhibition of HIV-1 in cell culture by oligonucleotide-loaded nanoparticles.

PURPOSE: To investigate the potential use of polymeric nanoparticles for the delivery of antisense oligonucleotides in HIV-1-infected cell cultures. METHODS: Phosphorothioate oligonucleotides were encapsulated into poly (D,L-lactic acid) nanoparticles. Two models of infected cells were used to test the ability of nanoparticles to deliver them. HeLa P4-2 CD4+ cells, stably transfected with the beta-galactosidase reporter gene, were first used to evaluate the activity of the oligonucleotides on a single-round infection cycle. The acutely infected lymphoid CEM cells were then used to evaluate the inhibition of the viral production of HIV-1 by the oligonucleotides. RESULTS: The addition to infected CEM cells of nanoparticles containing gag antisense oligonucleotides in the nanomolar range led to strong inhibition of the viral production in a concentration-dependent manner. Similar results were previously observed in HeLa P4-2 CD4+ cells. Nanoparticle-entrapped random-order gag oligonucleotides had similar effects on reverse transcription. However, the reverse transcriptase activity of infected cells treated with nanomolar concentrations of free antisense and random oligonucleotides was not affected. CONCLUSIONS: These results suggest that poly (D,L-lactic acid) nanoparticles may have great potential as an efficient delivery system for oligonucleotides in HIV natural target cells, i.e., lymphocytic cells.

Cell Survival↗
PubMed2001

Evidence for higher-order structure formation by the c-myb 18-mer phosphorothioate antisense (codons 2-7) oligodeoxynucleotide: potential relationship to antisense c-myb inhibition.

We have demonstrated the formation of higher-order structures (presumably tetraplexes) by an 18-mer phosphorothioate antisense c-myb oligodeoxyribonucleotide that has been shown to have activity in the treatment of leukemia xenograft models. Although not observable by conventionally employed techniques, such as PAGE and dimethyl sulfate (DMS) protection, the formation of such higher-order structures by this oligonucleotide was revealed by several techniques. These included capillary gel electrophoresis (CGE), which demonstrated the presence of molecules with greatly increased retention time compared with the monomer; magnetic circular dichroism spectroscopy, which demonstrated a band at 290 nm, a characteristic of antiparallel tetraplexes; and fluorescence energy transfer measurements. For the last, the 18-mer phosphorothioate oligonucleotide was synthesized with a 5'-fluorescein group. Similar to the molecular beacon model, its fluorescence was quenched when combined in solution with tetraplex-forming oligomers that contained a 3'-Dabcyl moiety. 7-Deazaguanosine inhibits the formation of tetraplexes by eliminated Hoogsteen base pair interactions. The wild-type and 7-deazaguanosine-substituted antisense c-myb oligomers differentially downregulated the expression of the c-myb proto-oncogene in K562 and HL60 cells, with the wild-type oligomer being the least active. The 18-mer c-myb molecule can, therefore, form highly complex structures, whose analysis in solution cannot be limited to examination of slab gel electrophoresis results alone.

Circular Dichroism↗
PubMed2001

Inhibition of potentially anti-apoptotic proteins by antisense protein kinase C-alpha (Isis 3521) and antisense bcl-2 (G3139) phosphorothioate oligodeoxynucleotides: relationship to the decreased viability of T24 bladder and PC3 prostate cancer cells.

Isis 3521 and G3139 are 20- and 18-mer phosphorothioate oligonucleotides, respectively, targeted to the protein kinase C (PKC)-alpha and bcl-2 mRNAs. Treatment of T24 bladder and PC3 prostate carcinoma cells with full-length and 3'-truncation mutants of Isis 3521 causes down-regulation of PKC-alpha protein and mRNA. However, at the level of a 15-mer and shorter, down-regulation of mRNA expression is no longer observed. Further, no diminution in cellular viability, as measured by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide assay, in response to increasing concentrations of paclitaxel, can be observed for these shorter oligomers. These observations not only indicate that PKC-alpha protein expression can be down-regulated by both RNase H-dependent and -independent mechanisms but also that down-regulation of PKC-alpha is insufficient by itself to "chemosensitize" cells. G3139, which down-regulates bcl-2 protein and mRNA expression, also down-regulates PKC-alpha protein and mRNA expression but not that of PKC-betaI, -epsilon, or -zeta. However, the down-regulation of PKC-alpha and bcl-2 are not linked. When the carrier Eufectin 5 is employed, only bcl-2 is down-regulated in both T24 and PC3 cells at 50 nM oligonucleotide concentration. At 100 nM, both bcl-2 and PKC-alpha expression are down-regulated, and only at this concentration can "chemosensitization" to paclitaxel and carboplatin be observed. In contrast, the down-regulation of bcl-2 seems to be linked with that of RelA (p65). However, this too is also not sufficient for chemosensitization, even though it leads to the loss of expression of genes under the putative control of nuclear factor-kappaB and to detachment of the cells from plastic surfaces. These results underscore the complexity of the intracellular requirements for the initiation of chemosensitization to anti-neoplastic agents.

Antineoplastic Agents, Phytogenic↗
PubMed2001

Antisense oligonucleotides: promise and reality.

Antisense oligonucleotides have been used for more than a decade to downregulate gene expression. Phosphodiester oligonucleotides are nuclease sensitive, and the more nuclease-resistant phosphorothioate oligonucleotides are now in common use in the laboratory and have entered clinical trials. However, these molecules are highly bioactive and may inhibit gene expression by more than one mechanism. Although some dramatic successes have been demonstrated, it can still be difficult to properly interpret experimental data derived from the use of this class of oligonucleotide. This review discusses some of these issues with particular reference to a major area of current interest--inhibition of bcl-2 expression in tumor cells.

Animals↗
PubMed2001

Chemosensitization of bladder carcinoma cells by bcl-xL antisense oligonucleotides.

PURPOSE: We investigated antisense inhibition of anti-apoptotic bcl-xL and bcl-2 proteins to increase chemosensitization in the T24 and 5637 bladder carcinoma cell lines. MATERIALS AND METHODS: A T24 bladder carcinoma cell line stably over expressing bcl-xL protein was constructed. Apoptosis by cytotoxic agents was estimated by cell cycle analysis and Annexin V binding. To eliminate bcl-xL expression T24 and 5637 cells were treated with C5-propynylated and 2'-O-methylribo-oligonucleotides. Levels of protein and messenger RNA were measured by Western and Northern blot analysis. Cell viability after combined treatment with oligonucleotides and various cytotoxic agents was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and evaluated statistically by Student's 2-sample t test. RESULTS: Forced over expression of bcl-xL protein desensitized the T24 bladder carcinoma cell line to cytotoxic agents. C5-propynylated and 2'-O-methylribo-oligonucleotides down-regulated bcl-xL protein expression in the T24 and 5637 cell lines, and increased their sensitivity to cytotoxic agents. The efficiency of antisense down-regulation of bcl-xL protein expression depended on the type of delivery agent. CONCLUSIONS: Antisense down-regulation of bcl-xL protein sensitizes bladder carcinoma cells to cytotoxic agents. However, it is possible that cellular chemosensitization results from a combination of effects, including nonsequence specificity, irrelevant cleavage and effects of the carriers combined with the specific antisense effects.

Apoptosis↗
PubMed2000

Bcl-xL in prostate cancer cells: effects of overexpression and down-regulation on chemosensitivity.

Both Bcl-xL and Bcl-2, antiapoptotic members of the Bcl family, are found in prostate cancer cell lines. Although these proteins may have similar antiapoptotic functions, it is not clear to what extent each serves as an antiapoptotic effector in prostate cancer cells. We engineered LNCaP and PC-3 cells to overexpress Bcl-xL protein and demonstrated that this desensitized them to the effects of cytotoxic chemotherapy. We then used two "antisense" strategies to down-regulate Bcl-xL protein expression in the parental lines. The first strategy used CS-propynylated phosphorothioate-phosphodiester oligonucleotides and co-down-regulated both Bcl-xL and Bcl-2; the second strategy used isosequential "gap-mer" phosphorothioate oligonucleotides containing 2'-O-methyl oligoribonucleotides at the 3' and 5' termini. In this case, only Bcl-xL protein expression was affected. The most active oligonucleotides of both types decreased the level of Bcl-xL protein expression to 5-30% of the control level. Multiple controls were inactive. Experiments combining oligonucleotide treatment with cytotoxic chemotherapeutic agents (paclitaxel, docetaxel, etoposide, vinblastine, carboplatin, and mitoxantrone) demonstrated a marked increase in the sensitivity of these prostate cancer cells. However, the increase in chemosensitivity in PC-3 cells was statistically identical (except mitoxantrone) for both "antisense" strategies, indicating that basal expression of Bcl-2, in contrast to that of Bcl-xL, may play little cytoprotective role in these cells.

Antineoplastic Agents↗
PubMed2000

Is irrelevant cleavage the price of antisense efficacy?

Antisense oligonucleotides are useful reagents for the suppression of gene expression. Their mechanism of action in eukaryotic cells appears to depend heavily on the activity of RNase H, a ubiquitous enzyme that cleaves the mRNA strand of an RNA-DNA duplex. However, the stringency requirements of RNase H are very low, and as little as a 5-base complementary region of oligomer to target may be sufficient to elicit RNase H activity. This would result in scission of nontargeted mRNAs, or what is known as "irrelevant cleavage." One strategy to reduce RNase H competency that has been employed is modification of the oligonucleotide backbone, replacing phosphodiester linkages with uncharged methylphosphonates, which are not RNase H competent. Another strategy involves replacement of deoxyribonucleic acid with 2'-O-alkylribonucleic acid. A third strategy, eliminating RNase H dependency entirely, requires activation of RNase P. The relative merits of these strategies will be discussed in the context of selective inhibition of gene function.

DNA↗