Increased prostate-specific membrane antigen expression in LNCaP cells following treatment with bispecific antisense oligonucleotides directed against bcl-2 and EGFR

Antisense oligonucleotides have been employed against in vivo and in vitro prostate cancer models. While most oligos consist of a single mRNA binding site, targeting a single gene product or others sharing sequence homology, our laboratory has developed bispecific oligos directed toward even unrelated proteins. This study evaluates the inhibition of in vitro propagating LNCaP cells employing mono- and bispecific oligos directed against bcl-2 [the second bispecific binding site was directed against the epidermal growth factor receptor (EGFR)]. Employing RT-PCR, the expression of non-targeted proteins encoded by mRNA for prostate-specific membrane antigen (PSMA) and prostate-specific antigen (PSA) were subsequently evaluated. When LNCaP prostate tumor cells were incubated with bispecific oligos (directed against bcl-2 and EGFR) and compared to lipofectin-containing controls significant growth inhibition resulted. In subsequent experiments, the levels of mRNA encoding PSMA were unexpectedly found to be elevated following treatment with the bispecific oligos but not with the monospecific directed solely against bcl-2. No differences were detected in mRNA levels encoding PSA following treatment with either mono- or bispecific oligos. Previously, we suggested that cell growth inhibition produced by some bispecifics could be attributed to complementary double-stranded regions formed by intra-strand base pairs. Double-stranded nucleic acids are known inducers of interferon, which promote expression of cell surface HLA type antigens. If induced, perhaps this cytokine also enhances PSMA expression, making prostate tumor cells a more recognizable target for cytotoxic T cells.     

Bax expression remains unchanged following antisense treatment directed against BCL-2

Antisense oligonucleotides (oligos) have been evaluated in both in vivo and in vitro prostate cancer models. Although most contain a single mRNA binding site, our laboratory has also evaluated bispecific types directed toward two proteins. This study evaluates the inhibition of in vitro propagating LNCaP cells employing mono- and bispecific oligos directed against bcl-2 [the second binding site was directed against the epidermal growth factor receptor (EGFR)]. Employing RT–PCR, the expression of two apoptosis regulating proteins, bcl-2 and non-targeted bax, was then evaluated. LNCaP prostate tumor cells were initially incubated for 24 h in the presence of oligos (6.25 μM) directed against bcl-2 and compared to lipofectin containing controls. Comparable and significant growth inhibition was produced by both mono- and bispecific forms. Employing RT–PCR to determine the expression of bcl-2, we found that the greatest amount of mRNA suppression approached 100% for each oligo type: monospecific MR₄ (directed only against bcl-2), 100%; and bispecifics MR₂₄ and MR₄₂, 86 and 100%, respectively. We conclude, based upon both inhibition of in vitro growth and bcl-2 expression, that bispecific antisense oligos directed against EGFR and bcl-2 mRNAs are at least as effective as a monospecific directed solely toward bcl-2. In an effort to determine a compensatory response by cells evading apoptosis in the presence of bcl-2 suppression, the levels of mRNA encoding the non-targeted apoptosis activating protein bax were evaluated. Non-targeted protein suppression by these bispecifics has previously been demonstrated against prostate-specific membrane antigen (PSMA). However, in contrast to effects against bcl-2 and PSMA, no significant alteration in bax expression was produced by either oligo type. In LNCaP cells, bcl-2 suppression does not influence bax expression and, at least for this protein, there is no compensatory change in bax expression regulating apoptosis at this level. Identifying changes in the expression of proteins which regulate apoptosis is important if gene therapy targets bcl-2.     

Compensatory and non-compensatory effects on protein expression following BCL-2 suppression by antisense oligonucleotides

Antisense oligonucleotides (oligos) have been employed against in vivo and in vitro prostate cancer models targeting growth regulatory proteins. In LNCaP cells, we evaluated both monospecific and bispecific oligos that targeted and comparably suppressed the expression of bcl-2, an apoptosis inhibitory protein. Cells compensated with both suppressed caspase-3 (an apoptosis promoter) activity, and an enhancement of both androgen receptor (AR) and p300 expression. This suggests that a progression to increased androgen sensitivity accompanies bcl-2 suppression, in this tumor line. To further evaluate mechanisms of adaptation, we now evaluate the effects upon the expression of insulin-like growth factor (IGF1) and another AR coactivator, IL-4, thought to increase prostate cancer growth. IGF1 expression was not significantly altered suggesting this pathway need not be regulated when bcl-2 directed gene therapy is employed. In contrast to increased AR and p300 expression that compensated for bcl-2 suppression, the AR coactivator IL-4 expression was not increased, suggesting no role in any increased androgen sensitivity.     

Combination chemotherapy employing bispecific antisense oligonucleotides having binding sites directed against an autocrine regulated growth pathway and bcl-2 for the treatment of prostate tumors

In previous studies we demonstrated that antisense oligonucleotides (oligos) against transforming growth factor-alpha (TGF-α [MR₁]), its binding site the epidermal growth factor receptor (EGFR [MR₂]), and the anti-apoptosis protein bcl-2 (MR₄) are efficacious against prostate tumors. In recent reports we also describe how two of these mRNA directed binding sites can be synthesized sequentially within a single linear complementary strand and administered either in the presence or absence of additional therapeutic agents. In these continuing experiments “bispecific” oligo pairs were further evaluated in the presence or absence of Cytoxan, Taxol, or DES. One oligo pair recognized the binding sites for TGF-α and EGFR mRNA (TGF-α/EGFR [MR₁₂] and EGFR/TGF-α [MR₂₁]); another pair recognized binding sites for EGFR and bcl-2 (EGFR/bcl-2 [MR₂₄] and bcl-2/EGFR [MR₄₂]). Oligo pairs differ in their linear 5′ to 3′ binding site orientations, and were tested in vitro against PC-3 and LNCaP prostate tumor cell lines. Following cell attachment, incubations were for 2 days with the agents followed by 2 days in their absence. When tested against PC-3 cells and combined with LD₅₀ Cytoxan, MR₂, MR₄, MR₂₄, MR₄₂ significantly inhibited 47.3, 45.7, 68.3, and 64.9%; with LD₅₀ Taxol MR₂, MR₄, MR₂₄, MR₄₂ significantly inhibited 49.8, 45.8, 64.1, and 59.2%; and with LD₅₀ DES MR₂, MR₄, MR₂₄, MR₄₂ significantly inhibited 66.6, 67.6, 64.3, and 67.2% respectively. Each agent significantly increased the inhibition produced by either oligo alone. LNCaP cells were also incubated with mono- and bispecific oligos in either the presence or absence of chemotherapeutics. MR₂, MR₄, MR₂₄, MR₄₂ produced significant inhibitions of 57.4, 58.4, 69.4, and 68.6% with LD₅₀ Cytoxan; 70.4, 70.1, 73.6, and 74.0% with LD₅₀ Taxol; and 49.8, 50.1, 59.6, and 53.9%, respectively with LD₅₀ DES. A complete PC-3 experiment compared MR₁, MR₂, MR₄, MR₁₂, MR₂₁, MR₂₄ and MR₄₂, in the presence of LD₅₀ Cytoxan. Each oligo combined with Cytoxan significantly inhibited: MR₁ by 51.0, MR₂ by 55.0, MR₄ by 58.0; MR₁₂ by 56.0; MR₂₁ by 61.1, MR₂₄ by 65.5 and MR₄₂ by 66.0%. Bispecifics directed against two different pathways, MR_24, and MR₄₂ were the most effective. A complete LNCaP experiment compared the same series of oligos also in the presence of LD₅₀ Cytoxan. Each oligo combined with Cytoxan significantly inhibited: MR₁ by 49.0, MR₂ by 50.0, MR₄ by 53.0; MR₁₂ by 52.0; MR₂₁ by 58.6, MR₂₄ by 53.9 and MR₄₂ by 58.0%.     

Treatment of MCF-7 breast cancer cells employing mono- and bispecific antisense oligonucleotides having binding specificity toward proteins associated with autocrine regulated growth and BCL-2

Antisense oligonucleotides (oligos) against transforming growth factor-alpha (TGF-alpha) (MR1) and its binding site, the epidermal growth factor receptor (EGFR) (MR2), are efficacious against the UACC 897 breast, PC-3 and LNCaP prostate, and T98G glioblastoma tumor lines in both in vitro and in vivo studies. Oligos against the anti-apoptosis protein bcl-2 (MR4) are also efficient against PC-3 and LNCaP tumors in similar in vitro experiments. To enhance activity, and also to introduce a derivative type of multifunctional oligo into this field, "bispecifics" were constructed containing two truncated complementary DNA sequences (from either MR1 or MR2) designed to bind targeted mRNA about their respective AUG initiation codons, and/or a similar sequence adjacent to the AUG site of mRNA encoding bcl-2. Tandem pairs of bispecifics were constructed: The first had complementary sequences for TGF-alpha and EGFR mRNA, but differed in 5' to 3' tandem orientation (TGF-alpha/EGFR [MR12] and EGFR/TGF-alpha [MR21] sequences); a second pair had binding sites associated with EGFR and bcl-2, also differing in orientation (EGFR/bcl-2 [MR24] and bcl-2/EGFR [MR42]). In studies targeting PC-3 and LNCaP cells, bispecifics demonstrated significant in vitro activity, and the second pair was significantly better than the original monospecifics. These studies are now extended to the MCF-7 breast cancer model in order to determine whether these particular bispecifics have similar anti-breast cancer activity and if they are significantly better than monospecific oligos from which they were derived. We conclude that bispecific oligos significantly inhibit MCF-7 growth, however, in contrast to results obtained with PC-3 and LNCaP, the monospecific oligos directed against EGFR and bcl-2 have significantly greater activity than the bispecifics targeting a combination of TGF-alpha, EGFR, or bcl-2. These data suggest that the relative activities of oligos, whether mono- or bispecific, change with tumor type. Bispecific oligos which target different proteins, possibly those which regulate estrogen utilization, may be more effective against MCF-7 cells and warrant additional investigation, particularly if co-administered with traditional chemotherapeutics.     

Inhibition of the EGFR with nanoparticles encapsulating antisense oligonucleotides of the EGFR enhances radiosensitivity in SCCVII cells

The aim of this study is to evaluate the effects of antisense epidermal growth factor receptor (EGFR) nanoparticles on cell survival and radiosensitivity in the head and neck squamous cell carcinoma cell line SCCVII. Experiments were performed using the murine head-and-neck tumor cell line, SCCVII. Nanoparticle encapsulated antisense EGFR oligonucleotides were combined with radiotherapy and the relative radiosensitivity of the cells was assessed in vitro by MTT and standard colony formation. The proportion of apoptotic cells and cell cycle stages were analyzed by flow cytometry. C3H/He mice with SCCVII tumor heterografts were treated with antisense-EGFR-nanoparticles or RT alone, or with combinations of concomitant and sequential therapy. The relative radiosensitivity of the tumors was assessed in vivo by growth delay assays. The SCCVII cells were resistant to anti-EGFR nanoparticles or radiation therapy alone, but a synergic inhibition effect was observed when the therapies were combined. When the SCCVII cells were pre-treated with 2 μg of antisense-EGFR nanoparticles for 24 h and X-irradiated (4 Gy), flow cytometry analysis revealed cell cycle arrest in G₁ phase and an increased proportion of apoptotic cells. Our results show that antisense EGFR nanoparticles enhance radiosensitivity by inhibition of EGFR-mediated mechanisms of radioresistance. Collectively, these findings may have therapeutic implications because EGFR inhibition may improve the therapeutic efficacy of radiation even in the tumor cells that are resistant to anti-EGFR therapy.     

BCL-2 antisense oligonucleotide genasense is active against imatinib-resistant BCR-ABL-positive cells.

PURPOSE: The near-universal emergence of imatinib resistance in patients with acute forms of Philadelphia chromosome-positive leukemia highlights the need for additional therapy to control this disease. G3139 (Genasense, oblimersen; Genta Inc.), a Bcl-2 antisense oligonucleotide, has been shown to down-regulate the Bcl-2 protein and induce apoptosis in myeloid leukemia cells from treated patients. We tested G3139 for its ability to inhibit BCR-ABL-mediated transformation in mice. EXPERIMENTAL DESIGN: Nude mice (n = 5/group) were transplanted s.c. with imatinib-resistant BCR-ABL-transformed TF-1 cells (BCR-ABL-TF-1-R cells). Mice with established tumors (0.1 g) were treated for 14 days with G3139 (7 mg/kg/day i.p.), or with the reverse-sequence control oligonucleotide G3622 (7 mg/kg/day i.p.) or with imatinib (50 mg/kg/day i.p.). RESULTS: Mice treated with G3622 or imatinib died within 10-12 weeks. Nearly all of the mice treated with G3139 survived for >6 months and had reduced tumor volume. Three of the 5 mice showed complete tumor regression. A transient decrease in Bcl-2 protein was observed that correlated with histological evidence of apoptosis. In addition, we harvested BCR-ABL-TF-1-R tumor cells from mice treated with G3139 or control G3622 (7 mg/kg/day i.p., 7 days). Cells were then cultured with the antileukemic agents imatinib, daunorubicin, 1-beta-D-arabinofuranosylcytosine, or etoposide. G3139 pretreatment resulted in enhanced induction of apoptosis by all of the agents. CONCLUSION: These results suggest that G3139 is a promising candidate for treatment of patients with imatinib-resistant Ph-positive leukemia, and that combination of G3139 and imatinib may be useful to circumvent clinically acquired imatinib resistance.     

Trastuzumab down-regulates Bcl-2 expression and potentiates apoptosis induction by Bcl-2/Bcl-XL bispecific antisense oligonucleotides in HER-2 gene--amplified breast cancer cells.

PURPOSE: To investigate the possible existence of an antiapoptotic cross-talk between HER-2 and antiapoptotic Bcl-2 family members. EXPERIMENTAL DESIGN: Bcl-2 and Bcl-XL expression and apoptosis induction were analyzed in HER-2 gene-amplified (BT474) and nonamplified (ZR 75-1) breast cancer cell lines exposed to trastuzumab, alone or in combination with either Bcl-2/Bcl-XL bispecific antisense oligonucleotides (AS-4625) or the small-molecule Bcl-2 antagonist HA14-1. RESULTS: In addition to HER-2 and epidermal growth factor receptor, trastuzumab down-regulated Bcl-2, but not Bcl-XL, protein, and mRNA expression in BT474 cells. Interestingly, trastuzumab-induced down-regulation of HER-2 and Bcl-2 was also observed in three of five and two of three breast cancer patients undergoing trastuzumab treatment, respectively. Despite Bcl-2 down-regulation, however, trastuzumab only marginally increased the rate of apoptosis (7.3 +/- 3.5%). We therefore investigated whether a combination of AS-4625 and trastuzumab might increase proapoptotic efficiency. AS-4625 treatment of BT474 cells decreased both Bcl-2 and Bcl-XL expression, resulting in a 21 +/- 7% net apoptosis induction; the combination of AS-4625 followed by trastuzumab resulted in a significantly stronger induction of apoptosis (37 +/- 6%, P <0.01) that was not observed with the reverse treatment sequence (trastuzumab followed by AS-4625). Similar results were obtained with the Bcl-2 antagonist HA14-1; indeed, exposure of BT474 cells to HA14-1 followed by trastuzumab resulted in a striking proapoptotic synergism (combination index=0.58 +/- 0.18), as assessed by isobologram analysis. CONCLUSIONS: Altogether our findings suggest that combined targeting of HER-2 and Bcl-2 may represent a novel, rational approach to more effective breast cancer therapy.     

Bispecific antisense oligonucleotides having binding sites directed against an autocrine regulated growth pathway and bcl-2 for the treatment of prostate tumors

Antisense oligonucleotides (oligos) against transforming growth factor-alpha (TGF-α) (MR₁) and its binding site, the epidermal growth factor receptor (EGFR) (MR₂), are efficacious against PC-3 and LNCaP prostate tumors. To enhance activity and aid in simultaneous delivery, “bispecific” 39-mer oligos were constructed containing portions of both MR₁ and MR₂ sequences. The first pair contained truncated sequences recognizing TGF-α and EGFR mRNA binding sites, about their respective AUG initiation codons. These bispecifics differ in their 5’ to 3’ tandem orientation (TGF-α/EGFR [MR₁2] and EGFR/TGF-α [MR₂₁] sequences). A second pair was constructed having complementary sequences for EGFR and bcl-2 (EGFR/bcl-2 [MR₂₄] and bcl-2/EGFR [MR₄₂]). All bispecifics were tested in vitro against PC-3 and LNCaP prostate tumor cells, and compared to mono-specific oligos from which they were derived. The purpose of this study was: (1) to evaluate bispecific antitumor activity; (2) to identify dominant sequences; (3) to identify effects of binding site orientation; and (4) to determine whether bispecifics are more effective when targeting one versus different growth-dependent pathways. Comparisons were made between oligos tested against either PC-3 or LNCaP cells incubated for 2 d with the agents followed by 2 d in their absence. The first PC-3 cell experiment demonstrated that bispecific MR₁2 and MR₂₁ oligos are at least as effective as their mono-specific counterparts and that the MR₂₁ bispecific orientation is more effective than the MR₁ mono-specific by 64% (p = 0.014). It also suggested that the sequence directed against EGFR contributed most to bispecific activity, particularly in the MR₂₁ orientation. In a second PC-3 study a second bispecific pair of 37-mer oligos was constructed containing bases complementary to mRNA encoding EGFR and the apoptosis-associated protein bcl-2 (MR₄). MR₂₄ was constructed with the EGFR complementary site at the 5’ end (EGFR/bcl-2), and MR₄₂, containing the opposite orientation (bcl-2/EGFR). Each contained the dominant EGFR activity identified previously. MR₁, MR₂, MR₄, MR₁2, MR₂1, MR₂4, and MR₄₂ (1X and 2X in concentration) were cultured with cells and compared to controls. Each oligo significantly inhibited growth of PC-3 cells. MR₄₂ was most effective and significantly better than MR₁ (p = 0.0128), MR₂ (p = 0.021), MR₄ (p = 0.0002), and MR₁2 (p = 0.0032). 2X MR₂4 and 2X MR₄₂ were better than their 1X concentration counterparts, but the differences were not significant. In a similar experiment MR₁, MR₂, MR4, MR₁2, MR₂1, MR₂4, and MR42 were cultured with LNCaP cells and compared to lipofectin-containing controls. Each oligo significantly inhibited the growth of LNCaP cells. Again, MR₄₂ was most effective and significantly better than MR₂ (p = 0.021) and MR₄ (p = 0.038). MR₂4 was significantly better than MR₂ (p = 0.048). Bispecific oligos are a significant advance in antisense technology and could play a role in treating prostate cancer, particularly if combined with traditional chemotherapeutics.     

Eradication of human non-Hodgkin's lymphoma in SCID mice by BCL-2 antisense oligonucleotides combined with low-dose cyclophosphamide.

Cancers overexpressing Bcl-2 protein, which prevents programmed cell death (apoptosis), are less sensitive to stresses that produce cellular damage, including chemotherapy. If the level of Bcl-2 protein can be reduced sufficiently using antisense oligonucleotides (ASOs) targeting the gene message, then cytotoxic agents may be rendered more effective in eliminating disease and increasing cure rate. Preclinical studies in SCID mice bearing Bcl-2 overexpressing systemic human B-cell lymphoma (DoHH2) were undertaken to support development of a clinical trial. These data confirm that a combination of an ASO (5 mg/kg) targeting bcl-2 and a low dose of cyclophosphamide (35 mg/kg) was an effective strategy, leading to the eradication of the DoHH2 cells in vivo and cure of the animals. When mice deficient in natural killer cell activity were treated with an ASO, similar results were observed, suggesting that ASO stimulation of the host immune system was not a significant factor in elimination of lymphoma cells. These studies indicate that therapeutic strategies involving the use of an ASO targeting bcl-2 in combination with a cytotoxic agent may improve clinical outcomes.     

Inhibition of LNCaP prostate tumor growth in vivo by an antisense oligonucleotide directed against the human androgen receptor

We have shown recently that a 15-mer phosphorothioate oligodeoxynucleotide (ODNas750/15) that hybridizes to the (CAG)n polyglutamine region of mRNA encoding human androgen receptor (AR) inhibits the expression of AR in LNCaP prostate cancer cells in vitro. This AR downregulation was accompanied by significant cell growth inhibition and reduced PSA secretion. In the present study we investigated the effects of this antisense AR ODN on prostate tumor growth in vivo using a mouse xenograft model. Via subcutaneously implanted diffusion pumps, either ODNas750/15 or a scrambled control sequence ODNsr750/15 was continuously administered into LNCaP tumor-bearing male nude mice for 7 weeks. Compared with untreated control animals, treatment with ODNas750/15 resulted in significant tumor growth inhibition. Retardation of tumor growth was also significant in castrated mice, whereas the scrambled control ODN did not exert any effects. No side effects such as loss of body weight were observed at any time of treatment. ODN treatment was well tolerated and, in contrast to castration, did not induce shrinkage of mouse prostates. Both AR expression in the tumor and PSA levels in mouse serum correlated with tumor size. However, we failed to demonstrate a correlation between tumor retardation and Ki-67 antigen expression and the number of apoptotic cells, respectively. Testing of antisense-treated LNCaP cells revealed that expression levels of other proteins that contain shorter polyglutamine sequence stretches such as HDAC2, TFIID, and c-jun were not affected. The present study demonstrates that downregulation of AR with antisense ODNas750/15 causes prostate tumor growth inhibition. These results further point out the important role of the AR in prostate tumors and support further testing of AR downregulation for treatment of prostate cancer.     

Lack of toxicity associated with the systemic administrat of antisense oligonucleotides for treatment of rats bearing LNCaP prostate tumors

Antisense oligonucleotides (oligos) are now in clinical trials for the treatment of a variety of diseases. However, concern is sometimes expressed as to the toxicity of such compounds, particularly those with phosphorothioated backbones. We have previously reported (J. Surg. Oncol. 62, 194, 1996) our experience in treating nude mice bearing human PC-3 prostate tumors with phosphorothioated antisense oligos directed against mRNA encoding transforming growth factor-alpha (TGF-α) and the epidermal growth factor receptor (EGFR). This therapy resulted in a 75% (9/12) response rate for the intralesional treatment and a 100% (3/3) response rate for the systemic administration utilizing Alzet diffusion pumps. In the current study, athymic nude rats bearing orthotopically implanted LNCaP tumors, whose establishment was confirmed by the expression of human PSA, were implanted subcutaneously with Alzet diffusion pumps and treated systemically for 14 days with a total of 1 mg of each cligo (2 mg total). Controls consisted of five untreated rats similarly inoculated with LNCaP cells, but which did not receive antisense oligos. After 2 weeks the rats were sacrificed and serum samples were evaluated for BUN, creatinine, LDH and SGOT. Lungs, kidneys, livers, spleens and prostates were also removed for pathologic evaluation. There were no serum marker differences between groups nor was there histologie evidence of oligo toxicity seen in any evaluated tissue. Of interest was the observation that the livers and spleens, as well as prostates, of treated animals revealed mild lymphocytic infiltration compared to controls. We conclude that at this level of administration, there is no toxicity associated with 14-day oligo treatment.     

Bcl-2/bcl-xL bispecific antisense treatment sensitizes breast carcinoma cells to doxorubicin,paclitaxel and cyclophosphamide

Overexpression of the anti-apoptotic proteins bcl-2 and bcl-xL is implicated in breast cancer development, tumor progression and drug resistance. Here we describe the use of the bcl-2/bcl-xL bispecific antisense oligonucleotide 4625 to sensitize breast carcinoma cells to anti-cancer drugs routinely used in breast cancer therapy. MCF7 cells were treated with oligonucleotide 4625, doxorubicin, paclitaxel or cyclophosphamide alone, or with combinations of oligonucleotide and the anti-cancer drugs. As measured in cell viability assays, treatment with the various combinations reduced the number of viable MCF7 cells more effectively than treatment with the single drugs alone. Treatment with a sequence control oligonucleotide did not affect cell viability. All combination treatments induced apoptosis as demonstrated by the appearance of massive nuclear condensation in a high proportion of the cells. To further characterize the interaction between 4625 and doxorubicin, paclitaxel or cyclophosphamide, the median-effect method was used. In MCF7 cells all combinations resulted in potent synergistic effects over a broad range of toxicity with combination indices ranging from 0.8 to 0.1. Similarly, strong synergistic interactions between oligonucleotide 4625 and the anti-cancer drugs were also observed in cultures of the breast carcinoma cell line MDA-MB-231. Our data suggest the use of 4625 as a potent adjuvant in breast cancer chemotherapy.     

Targeting of human breast cancer by a bispecific antibody directed against two tumour-associated antigens: ErbB-2 and carcinoembryonic antigen.

Carcinoembryonic antigen (CEA) and ErbB-2 are expressed in about 50 and 30% of breast cancers, respectively. We hypothesised that targeting of these two antigens by a bispecific antibody (BAb) might provide efficient tumour uptake and prolonged tumour residence time. In the present study, we first studied the expression of CEA and ErbB-2 on primary breast tumours screened by immunohistochemistry. Of 106 primary breast cancers, 69 (65%) were positive for CEA, 20 (19%) were positive for ErbB-2, and 13 (12%) expressed both antigens. We then prepared and evaluated a BAb directed against CEA and ErbB-2. Using BIACORE technology, we showed that the BAb recognised both CEA and ErbB-2 with affinities of 0.9 x 10 and 0.8 x 10 M(-1), respectively. In vivo, BAb tumour localisation was compared with that of its parental homodimeric F(ab')(2)-ORTHO-phenylene- dimaleimide (PDM) fragments. Uptake of (125)I-BAb was lower than that of (131)I-35A7F(ab')(2)-PDM in LS174T tumours, used as a model of CEA expressing tumours, and was similar to that of (131)I-FWP51 F(ab')(2)-PDM in SKOv3 tumours, used as a model of ErbB-2 expressing tumours. In a double-positive model, the SKOv3-CEA-1B9 tumour, BAb showed a similar uptake to that of 35A7 F(ab')(2)-PDM and we demonstrated that, although BAb had double specificity, it internalised as a homodimeric anti-ErbB-2 antibody. BAb showed a greater uptake than that of FWP51 F(ab')(2)-PDM and this difference was even more important 72 h after injection with an uptake of 7.3 +/- 2.1 vs. 1.4 +/- 0.5% of the injected dose per gram of tissue. The results obtained with the BAb in the double-positive tumour-bearing nude mice suggest that targeting two distinct tumour-associated antigens on the same cell could improve tumour localisation.     

Dihydrotestosterone interacts with EGFR/MAPK signalling and modulates EGFR levels in androgen receptor-positive LNCaP prostate cancer cells

Androgen receptor (AR) signalling plays a pivotal role in prostate cancer pathogenesis and progression. However, androgen-mediated AR signalling is yet to be fully understood. EGFR and MAP kinase signalling pathways play predominant roles in AR function. Therefore, we investigated the interaction of EGFR signalling and AR activity in AR-positive LNCaP cells. We found that 5alpha-dihydrotestosterone (DHT) and EGF had a synergistic effect on AR activity as detected by a luciferase reporter system, although EGF alone did not activate AR. Both ERK1/2 and p38 were involved in DHT and DHT/EGF-induced AR activation as detected by specific MEK and p38 inhibitors. Furthermore, 24-h treatment of the cells with DHT resulted in ubiquitination and down-regulation of the EGFR. This effect could be inhibited by the anti-androgen flutamide, suggesting an androgen-dependent mechanism. On the other hand, DHT-treatment strongly increased AR levels in LNCaP cells. These data suggest a complex regulatory loop between activated AR and EGFR. In conclusion, activation of AR by both DHT and EGF/DHT involves the MAP kinase pathway. Long-term activation of AR results in increase of AR levels, which through so far unknown regulatory mechanisms results in ubiquitination and degradation of the EGFR.     

Increase in cell surface antigen expression by leukemic cells following in vitro treatment with papain

A variety of enzymes have been used to increase the immunogenicity of tumor cells in syngeneic hosts, including neuraminidase, trypsin and papain. The results of the investigation presented here suggest that, under defined conditions, in vitro treatment by papain of living cells from a Gross virus induced leukemia results in a quantitative modification of the antigens expressed at the cell surface without any detectable qualitative modification. Cells from the W/Fu C58(NT)D leukemia (leukemia induced in W/Fu rats by the Gross agent) were submitted to a moderate in vitro treatment by papain. Antisera were raised in syngeneic W/Fu rats immunized with W/Fu C58 (NT)D cells, either untreated (G-typing serum) or papain treated. The cytotoxic activity of both of these types of antisera was assessed on W/Fu C58 (NT)D cells, untreated or papain treated. Target cells treated with papain showed an increased susceptibility to lysis by antibody in the presence of complement. Cross-absorption studies showed that the activity towards papain-treated target cells could be absorbed out with untreated W/Fu C58 (NT)D cells and was not affected by absorption with papain-treated normal lymphocytes.Hence papain treatment appears to induce an increase in antigen expression at the cell surface, rather than the unmasking of “cryptantigens”, and this could be of interest in producing antibodies against weak antigens.     

Novel strategy of liver cancer treatment with modified antisense oligonucleotides targeting human vasohibin-2

Vasohihibin-2 (VASH2) is a homolog of vasohibin-1 (VASH1) and is overexpressed in various cancers. Vasohihibin-2 acts on both cancer cells and cancer microenvironmental cells. Previous analyses have shown that VASH2 promotes cancer progression and abrogation of VASH2 results in significant anticancer effects. We therefore propose VASH2 to be a practical molecular target for cancer treatment. Modifications of antisense oligonucleotide (ASO) such as bridged nucleic acids (BNA)-based modification increases the specificity and stability of ASO, and are now applied to the development of a number of oligonucleotide-based drugs. Here we designed human VASH2-ASOs, selected an optimal one, and developed 2′,4′-BNA-based VASH2-ASO. When systemically administered, naked 2′,4′-BNA-based VASH2-ASO accumulated in the liver and showed its gene-silencing activity. We then examined the effect of 2′,4′-BNA-based VASH2-ASO in liver cancers. Intraperitoneal injection of naked 2′,4′-BNA-based VASH2-ASO exerted a potent antitumor effect on orthotopically inoculated human hepatocellular carcinoma cells. The same manipulation also showed potent antitumor activity on the splenic inoculation of human colon cancer cells for liver metastasis. These results provide a novel strategy for the treatment of primary as well as metastatic liver cancers by using modified ASOs targeting VASH2.     

Limited synergistic effect of antisense oligonucleotides against bcr-abl and transferrin receptor mRNA in leukemic cells in culture

The synergistic use of antisense oligonucleotides (ASOs) towards the bcr-abl and the transferrin receptor (TfR) mRNA was studied in a chronic myeloid leukemia (CML) cell line, aiming to improve the efficiency of individual ASO treatment. At 20 microM concentration, bcr-abl ASOs reduced cell growth by 40% and was specific for cells that have the translocation: there was a 34% reduction of BCR-ABL protein. The TfR ASO reduced cell growth by 20% and decreased TfR protein by 24%. The ASOs were more potent at reducing cell growth when used in combination (respectively, -20 and -17% than bcr-abl ASO and TfR ASO when used individually at the 10 microM concentration), thus we postulate that there is synergism of action. Cell cycle analysis also revealed that the sub-G1 peak was bigger in the synergistic treatment.