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Experimental Therapeutics, Preclinical Pharmacology |
Divisions of Medical Oncology [R. J. K.] and Pathology [R. D. G.] and Department of Advanced Therapeutics [R. J. K., M. B. B., R. N., J. H. G., F. M. P. W.], British Columbia Cancer Agency, Vancouver V5Z 4E6, and Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver V6T 2B5 [M. B. B., F. M. P. W.], British Columbia, Canada
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although elegant experiments have been carried out to define the mechanisms by which ASOs can specifically abrogate gene function, recent investigations have raised some puzzling questions regarding what factors govern their biological activity. First, ASOs can be very potent immune stimulators, by virtue of their unmethylated CpG motifs within the context of certain flanking sequences. Therapeutic activity can be attributed to systemic immune effects rather than to specific ASO/mRNA interactions (6, 7, 8, 9) . The development of control ODNs, containing similar backbone, codon length, and CpG motifs, has been useful to provide evidence for biological action linked to a specific ODN sequence. Second, for ASOs to be active delivery to the inside of a target cell must be achieved efficiently. In tissue culture, for example, this requires association with a delivery system such as cationic lipids (10 , 11) . In vivo, however, ASOs are active when given in free form (12, 13, 14) . Although studies assessing the mechanism of ASO activity in vivo are critically important to the further advancement of this technology, our primary concern remains the identification of agents with proven therapeutic activity in the clinical setting for disseminated systemic malignant disease.
An 18 mer phosphorothioated oligonucleotide, G3139, directed against the first six codons of the open reading frame of the bcl-2 gene message has been developed by Genta Inc. (Lexington, MA) and is used in our studies. The target protein, Bcl-2, is an antiapoptotic member of a large family of genes involved in the regulation of programmed cell death (15 , 16) . Studies of G3139 on the Bcl-2-overexpressing lymphoma cell lines DoHH2 and SU-DHL-4 in vitro have shown down-regulation of message and resultant decrease in protein expression (14) . Tumor xenograft models in SCID mice using G3139 alone have demonstrated therapeutic activity that is specific when compared with control antisense sequences (3 , 14) . Pharmacokinetic as well as toxicity studies have been performed identifying a dose range with a good therapeutic index (17 , 18) . Used as a single agent in a Phase I study in patients with relapsed NHL G3139 has been reported to modify Bcl-2 levels in clinical samples of lymphoid cells using doses that were also associated with objective responses (19) .
More recently, in vitro experiments have suggested that Bcl-2 plays a major role in the response of malignant cells to a variety of stresses that produce cellular damage, including chemotherapy (20, 21, 22) . Malignant cell lines transfected with the bcl-2 gene, with resultant overexpression of the protein product, demonstrate increased resistance to various chemotherapeutic agents (23, 24, 25, 26) . Additionally, cell lines overexpressing Bcl-2 are rendered more sensitive to killing by chemotherapeutic agents, either with introduction of ASOs directed at the bcl-2 message into culture or on transfection of the cells with a vector bearing the antisense sequence (27 , 28) . This resultant chemosensitization has been correlated with down-regulation of Bcl-2 expression.
The studies reported here assess the in vivo therapeutic potential of combining ASOs targeting bcl-2 with a low dose of a cytotoxic agent commonly used in the treatment of lymphoma. It is believed that this chemosensitizing effect correlates with specific ASO-mediated down-regulation of bcl-2 mRNA message and, subsequently, Bcl-2 protein. This is supported by PCR data and immunohistochemical evaluation of bone marrow obtained from DoHH2-bearing mice. Furthermore, results in perforin-deficient mice demonstrate that elimination of lymphoma cells is not a result of natural killer cell function.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DoHH2 is an EBV-negative human B-cell NHL cell line that carries the t(14;18) and has been extensively studied (29) . Following the initial description, it was found that a complex translocation involving chromosomes 8, 14, and 18 resulted in a derivative 8, which contained both the c-myc and bcl-2 oncogenes juxtaposed to the immunoglobulin heavy chain region with resultant overexpression of both protein products (30) . DoHH2 cells were grown in RPMI media containing 5% horse serum and 5% fetal bovine serum with 10 units/mL and 0.1 mg/mL penicillin/streptomycin and 2 mM L-glutamine. Cells were used for experiments between passages 8 and 17.
Preparation of LUV/ODN Complexes.
Bcl-2 expression could not be down-regulated with ASOs in the absence
of a synthetic oligonucleotide delivery agent, as demonstrated by our
results and others (11
, 31)
. For this reason, cationic
liposomes consisting of DODAC and DOPE were used to enhance ASO
intracellular delivery. ODNs in sterile water were diluted to a 5
µM concentration. DODAC:DOPE LUVs (Inex Pharmaceuticals
Corp., Vancouver, British Columbia) were diluted in sterile water to a
concentration of 4.6 mM total lipid to an equal volume to
the ODNs solution. The LUVs had a mean diameter of 80 ± 20 nm
before the formation of LUV/ODN complexes, as determined by
quasi-elastic light scattering (Nicomp submicron particle sizer
operating with an argon laser at 675.8 nm). ODNs were combined with the
diluted LUV solution in one addition at 4°C, and LUV/ODN complexes
immediately formed as indicated by a change in sample turbidity. Final
charge ratio of ODN:cationic lipids was 1.3:1 (+/-), and final ODN
concentration was 2.5 µM. After incubation for at least
30 min, LUV/ODN complexes (200–400 nm using quasi-elastic light
scattering) were added to DoHH2 cells.
In vitro Delivery Assays.
DoHH2 cells were plated at a concentration of 1 x 106
cells/ml in 6-well tissue culture-treated plates. All cells were plated
in RPMI media in the absence of serum. Cells were incubated for 4 h at 37°C and, subsequently, RPMI media was replaced with 5% fetal
bovine and 5% horse serum-containing media. After 72 h, cell
lysates were obtained using 0.15% SDS in 50 mM Tris (pH
8.0) and the expression of Bcl-2 protein was determined using Western
blot analysis.
Western Blot.
Western blot analysis was completed as described previously
(32)
. Protein concentrations in the obtained cell lysates
were determined by absorbance at 280 nm. Approximately 75–100 µg of
protein were separated using polyacrylamide gel electrophoresis.
Western transfer was completed on nitrocellulose membrane and blocked
with 5% skim milk. Bcl-2 primary antibody was diluted to 12 ng/ml, and
ß-actin primary antibody (DAKO, Carpenteria, CA) was diluted to 10
ng/ml in Tris-buffered saline containing 1% skim milk and 0.1% Tween
20 for 1 h. Antimouse secondary antibody was diluted to 20 ng/ml
in buffer and incubated at room temperature for 30 min. Bands were
detected via chemiluminescence using enhanced
chemiluminescence reagent from Amersham (Baie D’Urfé, Quebec,
Canada). Membrane was exposed to X-Omat Kodak film (Mandel
Scientific, Guelph, Ontario, Canada) for 30 s-1 min.
In vivo Model.
Male SCID/Rag-2 mice used for these studies were obtained from a
breeding colony at our institution when they were 6–9 weeks of age,
weighed 22 g and were maintained in a pathogen-free environment.
Viable DoHH2 cells (5 x 106 cells in 200 µl) were
injected i.v. via the tail vein of each animal, and disease
was allowed to establish for 4 days. Cohorts of at least three animals
were then treated in the following groups: (a) untreated
control (injected with saline); (b) CPA (Carter-Horner Inc.,
Mississauga, Ontario, Canada) at 15, 35, 75, or 150 mg/kg i.p. on days
4, 8, and 12; (c) ASO, RPO, or MMO at 5.0 or 12.5 mg/kg QD
or QOD i.p. for 14 treatments; and (d) ASO, RPO, or MMO QD
or QOD i.p. for 14 treatments in combination with CPA i.p. on days 4,
8, and 12. Animals were assessed for illness by nonbiased technicians
and terminated at signs of illness including, but not limited to,
paralysis in the hind region, scruffy coat, lethargy, weight loss of
>20%, or if they survived past 90 days.
Pfp/Rag-2 male mice were obtained from Taconic (Germantown, NY). These are SCID/Rag-2 mice deficient in perforin synthesis. Although natural killer cells are present in these animals, they are not capable of lysing cells. Animals were used within 2 weeks of arrival at our facility. Cohorts of five or six animals received injections of 5 x 106 DoHH2 cells in 200 µl i.v. via the tail vein. On day 4 after tumor cell inoculation animals began treatment with ASO alone (5 mg/kg for 14 treatments QOD) or in combination with CPA (35 mg/kg; days 4, 8, and 12) injected i.p. Animals were assessed and terminated at signs of illness, as described above.
Immunohistochemistry.
Microscopic sections of a femur for controls or treated animals were
processed routinely for H&E staining and reviewed by an experienced
hematopathologist (R.D.G.). Paraffin section immunohistochemistry was
performed to analyze Bcl-2 expression, as described previously
(33)
.
Molecular Genetics.
High MW DNA was extracted from all tissue specimens and the DoHH2 cell
line with an automated DNA extractor (Applied Biosystems Model 341;
Perkin-Elmer Corp., Foster City, CA). PCR for immunoglobulin heavy
chain and the presence of a bcl-2 (mbr) rearrangement was
performed, as described previously (34)
. The presence of
amplifiable DNA in the reactions was confirmed in all cases by a
parallel amplification of a 155-bp and a 510-bp segment of the
p53 and ß-globin genes, respectively.
Statistical Analysis.
Cohorts were determined by combining several experiments together.
Survival analysis data were evaluated with censored regression, with
median survival times determined using the Cox’s f-test.
This was done because several of the treated groups consisted of
long-term survivors (>90 days). For these groups median survival times
were estimated based on the assumption that the surviving animals died
on day 91. Groups with >50% 90-day median survival were labeled not
obtained (NO). Cohorts with less than three uncensored data points were
labeled not determined (ND) because estimated parameters were not
reliable. Survival curves were computed using the Kaplan-Meier method.
Treatment groups were subsequently analyzed using Statistica software
and compared using a two-sample log-rank test. p-values were
derived from the log-rank test comparing two samples and were reported
if p < 0.05 or were determined to be not significant
(NS).
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RESULTS |
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. Cells were
treated with either ASO or two control ODNs consisting of a full-length
reverse-polarity sense sequence (RPO) as well as a two-base mismatch
sequence to G3139 (MMO). Of note, these control oligonucleotides
contain similar backbone, codon length, and CpG motifs as G3139. ODNs
were delivered in free form or with cationic liposomes (DODAC:DOPE
LUVs) as a carrier. The concentration of ODNs used (2.5
µM) was the minimum concentration, following complexation
with cationic lipids, that clearly demonstrated down-regulation of
Bcl-2 protein with minimal toxicity (data not shown). The cell toxicity
that was observed following ASO addition was not due to associated
lipids. DoHH2 cells expressed the Bcl-2 protein as indicated by the 26
kDa band, whereas delivery of LUV/ASO complexes specifically
down-regulated Bcl-2 protein expression. This effect was sequence
specific because it was not seen with the control oligonucleotides,
where results were indistinguishable from untreated cells. This
antisense effect correlated with a small decrease in cell number and
could only be obtained when using a formulation method involving ASO
complexation with cationic liposomes to engender ASO delivery.
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shows the
representative section through a femur in untreated control animals,
and Fig. 2B
shows the corresponding Bcl-2 immunoperoxidase
staining. Extensive infiltration with Bcl-2-expressing human lymphoma
is observed with complete replacement of murine marrow, and Bcl-2
immunoperoxidase staining is strikingly positive in control femur and
lymph nodes. Similarly, bcl-2 and immunoglobulin heavy chain
PCR demonstrated a single dominant band in the untreated control
animals of approximately 100 bp and 365 bp, respectively (Fig. 2E
, Lane 2).
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and 2
and
Fig. 3
, assessed a range of doses of both ODNs and CPA. Two dosing
schedules of ODNs were evaluated, each schedule consisting of 14 total
i.p. injections, one given over a period of 18 days and the other over
a period of 28 days. Dose scheduling was based on previous reports that
phosphorothioate ODNs have elimination half-lives of 
12–72 h
(17)
. CPA was given consistently i.p. 4, 8, and 12 days
after tumor cell injection. Untreated control animals, as well as the
RPO- and MMO-treated control groups, were terminated or died with
progressive tumor at a median of 33–37 days after tumor cell
inoculation (see Table 1
). No long-term survivors were observed in
these groups. CPA-treated animals (Table 2)
showed a dose-response
correlation with no effect (median survival time, 36 days) seen at a
dose of 15 mg/kg and modest effect (median survival time, 47 days) at
35 mg/kg. The DoHH2-bearing animals could be treated at higher dose of
CPA (75 and 150 mg/kg), and at these doses 100% long-term (>90 days)
survival was observed. Therefore, the two lower doses (15 and 35 mg/kg)
were selected for studies evaluating combinations with ASO.
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. In
comparison with untreated animals, those treated with even the lowest
dose of ASO showed a significant (p < 0.000001)
increase, when compared with control, in median survival from 33 days
to at least 62 days. Dose-dependent increases in therapeutic efficacy
were most easily observed by monitoring the percentage of long-term
(>90 days) survivors after treatment. As the dose increased from 2.5
to 5 to 12.5 mg/kg/day, the number of long-term survivors increased
from 17% to 48% to 58%, respectively. The differences in response
between the 5 and 12.5 mg/kg dose levels were not significant. If the
dosing schedule was changed to QD injections there was a slight, but
not significant, decrease in therapeutic activity noted. The QD
schedule at 12.5 mg/kg gave a long-term survival rate of 44% and a
median survival time of 85 days, whereas the QOD schedule resulted in a
long-term survival rate of 58%. There was no statistically significant
difference between results when animals were treated QD or QOD.
It is worth noting that pathological evaluation at necropsy was
completed in all long-term survivors and those animals showed no
evidence of tumor either on gross inspection or on histological
examination of tissues. Molecular studies, designed to detect the human
bcl-2 gene by PCR, failed to detect residual disease in
these mice (Fig. 2E
, Lane 4). In contrast, those animals
that were terminated during the course of these studies because of
disease progression, whether in an ASO or a CPA alone treatment group,
were found to have enlarged lymph nodes and tumor nodules consistent
with those of untreated mice, as well as molecular evidence of disease
as demonstrated by PCR (
, Lane 2).
Immunohistochemistry was used to confirm the presence of
bcl-2-expressing human lymphoma in the lymph nodes, spleen,
and femoral bone marrow. The findings established that the progression
of the human lymphoma was the cause of death in these animals. It was
concluded on the basis of these results that death during the 90-day
time course was always associated with progression of the DoHH2 tumor
and that animals surviving beyond 90 days were free of any residual
disease. Fig. 2,C and D
, shows a representative
femoral bone marrow of a combination treated animal (5.0 mg/kg ASO and
35.0 mg/kg CPA) that survived past 90 days. It was observed that there
was no human lymphoma cell infiltrate (Fig. 2C)
and Bcl-2
staining was negative (Fig. 2D)
. Regardless,
immunohistochemistry was routinely performed on samples from all
animals that survived or were terminated as a consequence of tumor
development. Some animals were maintained in the vivarium for time
periods in excess of 120 days. In addition, selected mice that survived
as a consequence of treatment received reinoculations of 5 x
106 DoHH2 cells to assess whether the SCID animals had
developed immune resistance to tumor cell challenge (results not
shown). These animals died within 35 days, typical of control animals,
and exhibited Bcl-2-expressing human lymphoma in spleen, lymph nodes,
bone marrow, and elsewhere.
It is clear, as assessed by immunohistochemistry and PCR, that ASO
treatment of lymphoma-bearing mice eliminated Bcl-2 expression
associated with the DoHH2 cell. This is most likely a consequence of
eradication of the tumor cells and may not be directly linked to
down-regulation of bcl-2 expression. It is possible that the
therapeutic activity is linked to ASO-induced immune stimulation and
tumor cell loss by activated killer cells. Reports from many
investigators have suggested that the mechanism of action for ASOs may
be, in fact, a result of their potency as immune stimulators rather
than specific down-regulation of protein expression
(6, 7, 8, 9)
. Although the SCID/Rag-2 mice used in these studies
were deficient in B- and T-cell maturation, we further examined the
effect of ASOs on immunostimulation by treating lymphoma-bearing mice
deficient also in perforin production (Fig. 4
and Table 3
). Pfp/Rag-2 mice received injections of
DoHH2 i.v. and were treated with ASO at 5 mg/kg alone for 14 treatments
(QOD) or in combination with CPA (35 mg/kg; three treatments, days 4,
8, and 12). Median survival of animals treated with ASOs increased from
27 days (control) to 37 days (P < 0.01). When mice
were treated with CPA in addition to ASO, median survival times
increased to 61 days when compared with animals treated with ASO only
(P < 0.01). Furthermore, 17% of the animals in the
combination-treated cohort survived past 90 days.
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and Fig. 3B
. The benefits
achieved by using the bcl-2 ASOs in combination with CPA are
best illustrated at the lowest doses. In the absence of ASOs, a dose of
CPA at 15 mg/kg resulted in no measurable increase in the life span of
mice bearing the DoHH2 tumors. Alternatively, ASO given alone at a dose
of 2.5 mg/kg/injection increased median survival to 62 days, and one of
six animals survived beyond 90 days. In combination, the median
survival times were 72 and 84 days when treated with CPA at 15 mg/kg
and ASO at 2.5 mg/kg and 5 mg/kg, respectively. Both of these treatment
groups exhibited long-term survival rates of 50% or more.
A similar pattern was also seen in animals treated at the 35 mg/kg dose
of CPA. When animals were treated with 35 mg/kg CPA or ASO at 5
mg/kg/injection, they exhibited median survival times of 47 and 79
days, respectively, with no long-term survivors for CPA and 48% for
ASO. When treated with both drugs the median survival times could not
be determined because long-term survival exceeded 60% (range,
61–100%, depending on cohort), regardless of whether the ASO dose was
2.5 or 5 mg/kg/injection. CPA administered in combination with the
control RPOs and MMOs resulted in a median survival of 57 and 53 days,
respectively, and this was not significantly different than the
activity observed for CPA given alone (median survival of 47 days). It
is worth noting that long-term survival increased significantly when
CPA was combined with ASOs given by the less effective QD dosing
schedule. As summarized in Table 2
, mice treated with ASO at 5 and 12.5
mg/kg/injection (QD) in combination with a 35-mg/kg dose of CPA
exhibited long-term survival rates of 100% and 83%.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Low-dose chemotherapy with an ASO that is directed at bcl-2 has the potential to lower the amount of antineoplastic agent required to eliminate disease and, therefore, reduce the associated toxicity. It is important to note that down-regulation of the specific antisense target protein need only be transient to minimize the growth advantage of the tumor cell such that the effect of chemotherapy is maximal. We have demonstrated that G3139 shows specific down-regulation of Bcl-2 and enhances the activity of low doses of CPA in this model and in perforin-deficient mice. When cured SCID/Rag-2 mice were challenged with DoHH2 cells, all animals exhibited symptoms of Bcl-2-expressing lymphoma and died within 35 days, similar to control. This would indicate that ASO treatment did not stimulate a lasting immune response. Furthermore, the studies in the pfp/Rag-2 mice strongly suggest that immunostimulation is not a factor in eradication of lymphoma cells in vivo. Rather, specific ASO/mRNA interactions leading to down-regulation of Bcl-2 protein may, in fact, prime the tumor cells for death pathways (i.e., apoptosis), leading to chemosensitization. Follicular lymphomas overexpress Bcl-2 as a survival advantage, and, in many instances, this overexpression is the sole determinant keeping at least a percentage of the malignant cells from going down the apoptotic pathway. If the apoptotic barrier is relieved, these cells appear sufficiently damaged for death.
Our studies conclusively demonstrate that combination treatment with
CPA and ASO renders SCID/Rag-2 mice curable of human lymphoma in a
large fraction of the animals. The interaction between the two agents
shows dose-response correlations (Tables 1
and 2)
. For both doses of
CPA, increasing the dose of ASO from 2.5 to 5 mg/kg resulted in longer
median survivals and an overall increase in long-term survivors. A
rather striking result was achieved when a completely ineffective dose
of CPA (15 mg/kg; median survival 36 days and no long-term survivors)
was combined with a modestly effective dose of ASO (2.5 mg/kg; 61 day
median survival and 16% long-term survivors) to produce a 72-day
median survival and 50% long-term survivors. These results suggest
that chemotherapy at very modest doses could be made much more
effective with use of ASOs without increasing the toxicity to normal
tissues. Such an increase in the efficacy of currently available
chemotherapeutic agents could significantly alter the prognosis of a
large number of modestly to moderately sensitive human tumors,
resulting in improved clinical outcomes or increasing the potential for
cure.
The model has direct relevance to the clinical situation faced in NHL where patients typically present with what appears to be a chemotherapy-sensitive tumor at diagnosis that regresses only to recur within months to years after treatment. Clinically, the prevalence of Bcl-2 overexpression in NHL is high, consisting of 90% in follicular and mantle cell histologies and 50% of diffuse large cell disease. The DoHH2 cell line was derived from a follicular lymphoma carrying a t(14;18) that results in constitutive bcl-2 gene overexpression. The aggressive nature of the disease in this model is, however, more suggestive of a transformation to a higher-grade histology, a common event in follicular lymphoma. Indeed, a recent reexploration of the molecular and cytogenetic features of the cell line, using more sensitive detection techniques, has revealed a second translocation involving the c-myc oncogene with a resultant derivative chromosome 8 carrying t(8;14;18) (30) . We have just recently described this clinical phenomenon of double translocation and constitutive overexpression of both bcl-2 and c-myc in a subset of patients with small noncleaved cell (Burkitt-like) lymphoma, which represents a very aggressive form of the disease (36) .
The data presented here are the first to address the potential role of ASOs directed at the bcl-2 gene message in enhancing the therapeutic efficacy of a cytotoxic agent in NHL. In a model of human melanoma implanted s.c. in SCID mice, Jansen et al. (10) have shown chemosensitization to dacarbazine (DTIC) with ASOs directed at bcl-2, resulting in reduced tumor volumes. The present study, however, is the first example of successfully using this strategy to increase the actual cure rate of any systemically distributed metastatic malignancy in a xenograft model. Moreover, the data suggest that improved clinical outcomes could be achieved with standard, or even lower, doses of anticancer drugs when combined with ASOs, potentially impacting overall clinical tolerance and costs of care. As a single agent, G3139 has entered clinical testing and promising initial results have been published, with a number of responses seen, in addition to down-regulation of Bcl-2 protein in clinical samples from patients treated for NHL (19) . Based directly on our results, we have initiated Phase I/II clinical trials of chemotherapy in combination with G3139 in patients with NHL.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the National Cancer Institute of
Canada (to M. B. B.). F. M. P. W. is a recipient of a GREAT
Fellowship from the Science Council of British Columbia and a
studentship from the Cancer Research Society, Inc. 
2 To whom requests for reprints should be
addressed, at Division of Medical Oncology, British Columbia Cancer
Agency, 600 West 10th Avenue, Vancouver, British Columbia, Canada V5Z
4E6. Phone: (604) 877-6000, ext. 2730; Fax: (604) 877-0585; E-mail: rklasa{at}bccancer.bc.ca
3 The abbreviations used are: ASO, antisense
oligonucleotide; ODN, oligodeoxynucleotide; RPO, reverse-polarity ODN;
MMO, mismatch ODN; MW, molecular weight; LUV, large unilamellar
vesicle; DODAC,
N-N-dioleyl-N,N-dimethylammonium
chloride; DOPE,
1,2dioleoyl-sn-glycero-3-phosphoethanolamine; CPA,
cyclophosphamide; QD, every day treatment; QOD, every other day
treatment; SCID, severe combined immunodeficient; NHL, non-Hodgkin’s
lymphoma.
Received 11/ 2/99; revised 2/28/00; accepted 2/28/00.
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Y. Hu, G. Bebb, S. Tan, R. Ng, H. Yan, J. R. Sartor, L. D. Mayer, and M. B. Bally Antitumor Efficacy of Oblimersen Bcl-2 Antisense Oligonucleotide Alone and in Combination with Vinorelbine in Xenograft Models of Human Non-Small Cell Lung Cancer Clin. Cancer Res., November 15, 2004; 10(22): 7662 - 7670. [Abstract] [Full Text] [PDF]
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A. W. Tolcher, J. Kuhn, G. Schwartz, A. Patnaik, L. A. Hammond, I. Thompson, H. Fingert, D. Bushnell, S. Malik, J. Kreisberg, et al. A Phase I Pharmacokinetic and Biological Correlative Study of Oblimersen Sodium (Genasense, G3139), an Antisense Oligonucleotide to the Bcl-2 mRNA, and of Docetaxel in Patients with Hormone-Refractory Prostate Cancer Clin. Cancer Res., August 1, 2004; 10(15): 5048 - 5057. [Abstract] [Full Text] [PDF]
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C. Thallinger, M. F. Wolschek, H. Maierhofer, H. Skvara, H. Pehamberger, B. P. Monia, B. Jansen, V. Wacheck, and E. Selzer Mcl-1 Is a Novel Therapeutic Target for Human Sarcoma: Synergistic Inhibition of Human Sarcoma Xenotransplants by a Combination of Mcl-1 Antisense Oligonucleotides with Low-Dose Cyclophosphamide Clin. Cancer Res., June 15, 2004; 10(12): 4185 - 4191. [Abstract] [Full Text] [PDF]
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A. Raffo, J. C. Lai, C. A. Stein, P. Miller, S. Scaringe, A. Khvorova, and L. Benimetskaya Antisense RNA Down-Regulation of bcl-2 Expression in DU145 Prostate Cancer Cells Does Not Diminish the Cytostatic Effects of G3139 (Oblimersen) Clin. Cancer Res., May 1, 2004; 10(9): 3195 - 3206. [Abstract] [Full Text] [PDF]
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 C. M. Rudin, M. Kozloff, P. C. Hoffman, M. J. Edelman, R. Karnauskas, R. Tomek, L. Szeto, and E. E. Vokes Phase I Study of G3139, a bcl-2 Antisense Oligonucleotide, Combined With Carboplatin and Etoposide in Patients With Small-Cell Lung Cancer J. Clin. Oncol., March 15, 2004; 22(6): 1110 - 1117. [Abstract] [Full Text] [PDF]
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 D. J. Waxman and P. S. Schwartz Harnessing Apoptosis for Improved Anticancer Gene Therapy Cancer Res., December 15, 2003; 63(24): 8563 - 8572. [Abstract] [Full Text] [PDF]
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A. Ortega, P. Ferrer, J. Carretero, E. Obrador, M. Asensi, J. A. Pellicer, and J. M. Estrela Down-regulation of Glutathione and Bcl-2 Synthesis in Mouse B16 Melanoma Cells Avoids Their Survival during Interaction with the Vascular Endothelium J. Biol. Chem., October 10, 2003; 278(41): 39591 - 39599. [Abstract] [Full Text] [PDF]
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J. A. Shabbits, Y. Hu, and L. D. Mayer Tumor Chemosensitization Strategies Based on Apoptosis Manipulations Mol. Cancer Ther., August 1, 2003; 2(8): 805 - 813. [Full Text] [PDF]
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Y. Hu, G. Cherton-Horvat, V. Dragowska, S. Baird, R. G. Korneluk, J. P. Durkin, L. D. Mayer, and E. C. LaCasse Antisense Oligonucleotides Targeting XIAP Induce Apoptosis and Enhance Chemotherapeutic Activity against Human Lung Cancer Cells in Vitro and in Vivo Clin. Cancer Res., July 1, 2003; 9(7): 2826 - 2836. [Abstract] [Full Text] [PDF]
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R. Loomis, R. Carbone, M. Reiss, and J. Lacy Bcl-2 Antisense (G3139, Genasense) Enhances the in Vitro and in Vivo Response of Epstein-Barr Virus-associated Lymphoproliferative Disease to Rituximab Clin. Cancer Res., May 1, 2003; 9(5): 1931 - 1939. [Abstract] [Full Text] [PDF]
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 B. Jahrsdorfer, R. Jox, L. Muhlenhoff, K. Tschoep, A. Krug, S. Rothenfusser, G. Meinhardt, B. Emmerich, S. Endres, and G. Hartmann Modulation of malignant B cell activation and apoptosis by bcl-2 antisense ODN and immunostimulatory CpG ODN J. Leukoc. Biol., July 1, 2002; 72(1): 83 - 92. [Abstract] [Full Text] [PDF]
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R. Mohammad, Y. Abubakr, M. Dan, A. Aboukameel, C. Chow, A. Mohamed, N. Hamdy, and A. Al-Katib Bcl-2 Antisense Oligonucleotides Are Effective against Systemic but not Central Nervous System Disease in Severe Combined Immunodeficient Mice Bearing Human t(14;18) Follicular Lymphoma Clin. Cancer Res., April 1, 2002; 8(4): 1277 - 1283. [Abstract] [Full Text] [PDF]
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J. M. Vose, B. C.-H. Chiu, B. D. Cheson, J. Dancey, and J. Wright Update on Epidemiology and Therapeutics for Non-Hodgkin's Lymphoma Hematology, January 1, 2002; 2002(1): 241 - 262. [Abstract] [Full Text]
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V. Poulaki, N. Mitsiades, M. E. Romero, and M. Tsokos Fas-mediated Apoptosis in Neuroblastoma Requires Mitochondrial Activation and Is Inhibited by FLICE Inhibitor Protein and bcl-2 Cancer Res., June 1, 2001; 61(12): 4864 - 4872. [Abstract] [Full Text] [PDF]
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R. J. Klasa, A. F. List, and B. D. Cheson Rational Approaches to Design of Therapeutics Targeting Molecular Markers Hematology, January 1, 2001; 2001(1): 443 - 462. [Abstract] [Full Text] [PDF]
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), ASO (
), RPO (
), MMO (
), and CPA only (•).