Antisense p53 transduction leads to overexpression of bcl-2 and dexamethasone resistance in multiple myeloma

1. Introduction 

Multiple myeloma is a B-cell malignancy characterized by neoplastic proliferation of plasma cells [1], [2]. Several studies have indicated that multiple myeloma progresses through the accumulation of a variety of secondary genetic changes [3], [4]. Non-random cytogenetic changes affecting chromosome 11 or a deletion or translocation affecting chromosome 13 have been correlated with a poor prognosis [5]. At the molecular level various abnormalities have been reported including changes in the p53 gene [6]. A high frequency of point mutations in the conserved exons of the p53 gene was reported in human myeloma cell lines [7]; while in primary myeloma cells, four out of six patients with advanced disease had point mutations within exons 5 or 7 of the p53 gene [8]. Acquisition of p53 point mutations have been reported on progression to clinically more aggressive acute or leukemic forms of multiple myeloma from indolent disease [9]. These results suggest that p53 plays an important role in the progression of multiple myeloma to a more aggressive form.

In a model proposed by Vogelstein et al. [10], the wild-type p53 gene binds to p53 DNA binding sites and controls the expression of p53 regulated genes that, in turn, negatively regulate cell growth and division. Several groups have introduced wild-type p53 gene in to cancer cells showing growth inhibition or arrest [11], [12], [13], [14], [15]. Results from in vivo experiments in which wild-type p53 is over expressed in cancer cells have shown these cells to have decreased tumorigenicity when compared to the parent cells [16].

Conversely, suppression of wild-type or mutated form of p53 expression has been attempted by using antisense oligonucleotides complementary to the mRNA that codes for p53. Mukhopadhyay et al. [17] reported an increased growth rate and tumorigenesis in a non-small cell lung cancer cell line. However, Bayever et al [18] have reported irreversible inhibition of the proliferation of primary acute myeloid leukemia blast populations using antisense p53 oligonucleotides.

We have, in this report, studied changes in a multiple myeloma cell line following transduction with an antisense p53 construct. These transfected cells showed up-regulation of bcl-2 levels, decreased apoptosis and resistance to dexamethasone-induced cell kill.

2. Materials and methods 

2.2. Plasmids 

Plasmid pAAV/Ad was kindly provided by Dr. Samulski (University of North Carolina, Chapel Hill, North Carolina). Plasmid pWP8a and WP19 were a gift from Dr. Srivastava. Plasmid pP53as was constructed by isolating a 2.3kb human p53 cDNA fragment from pHP53B obtained from ATCC by digestion with BamHI and HindIII. The cDNA was cloned in an antisense orientation under the control of a herpes virus thymidine kinase promoter into the BamHI site of pWP19 vector which also contained neomycin-resistance gene (NeoR) as a selectable marker between AAV termini as shown in Fig. 1.

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Fig. 1. Recombinant AAV plasmids pWP8a containing neomycin phosphotransferase gene (NeoR) driven by herpes virus thymidine kinase promoter (Tkp) and pP53as containing p53 gene in antisense orientation driven by Tkp with the NeoR driven by TKp.

3. Results 

Following the transduction and G418 selection of the ARH77 cells, the rate of cell proliferation was measured by counting the number of live cells on alternate days. As seen in Fig. 2, the growth of cells transduced with only the NeoR gene, was similar to non-transduced cells. However, cells transduced with the antisense p53 (p53as) gene showed accelerated growth, as measured by the number of live cells. Fig. 2 represents mean of three culture assays performed on these cell lines. This accelerated growth was not associated with any appreciable change in the morphology of the cells (data not shown).

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Fig. 2. Cell growth curve representing number of trypan blue negative cells in culture over 8 days showing significantly faster cell growth by p53as-transduced cells compared to NeoR-transduced or untransduced cells.

To confirm a change in p53 expression by transduction of the antisense p53, we measured sense p53 RNA by an RNase protection assay using -labeled antisense RNA probe representing 118 bp of exon 7 of a p53 cDNA fragment as described above. As seen in Fig. 3 a marked suppression of sense p53 RNA was observed in the cell line transduced with the antisense construct compared to untransduced control cells or cells transduced with NeoR only. The p53 protein expression was also confirmed to be markedly suppressed as studied by immunohistochemistry (data not shown). To determine other changes that resulted in an increased growth rate, after the introduction of antisense p53 in the cell line, we investigated the expression of the genes coding for c-myc, bcl-2 and IL-6. All of these genes have been reported to be important in multiple myeloma growth or survival. A significant increase in the expression of bcl-2 in p53as-transduced cells was observed as compared to control NeoR-transduced cells (Fig. 4). The ratio of bcl-2/actin was 2.2 for NeoR-transduced cells compared to 18 for p53as-transduced cells. The expression of IL-6 and myc was similar both in control and p53as-transduced cultures (Fig. 5).

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Fig. 3. Autoradiogram of ribonuclease protection gel assaying for p53 mRNA expression in ARH77 cells. Lane 1 and 3 are control samples from untransduced and NeoR-transduced ARH77 cells; Lane 2 is p53as-transduced cells showing marked reduction in p53 sense RNA in p53as-transduced cells while p53 transcript is present in both control samples.

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Fig. 4. Northern blot analysis of bcl-2 gene expression. Total cellular RNA from untransduced, p53as-transduced and NeoR-transduced ARH77 cells was studied for levels of bcl-2 and β-actin. Increased expression of bcl-2 was noted in the p53as-transduced cells.

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Fig. 5. Northern blot analysis of c-myc and interleukin-6. Total cellular RNA from untransduced, p53as-transduced and NeoR-transduced ARH77 cells was studied for levels of c-myc, interleukin 6 and β-actin. No significant difference in expression of either gene was noticed between all the three cell lines.

In a variety of cell types, increased expression of bcl-2 correlates with a decrease in programmed cell death, with increased cell survival. We measured spontaneous apoptosis in the p53as-transduced cells. As seen in Fig. 6, 14.0±3.7% of the control cells underwent spontaneous apoptosis; however, there was a marked reduction in apoptosis in the p53as cells to 1.2±0.5%.

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Fig. 6. The p53as-transduced and control untransduced ARH77 cells were studied for spontaneous apoptosis by DNA end-labeling method. The p53as-transduced cells shows 1.2±0.5% apoptotic cells compared to 14.0±3.7% for control untransduced cells.

Next, we studied the responsiveness of these cells to killing by dexamethasone. Cells were exposed to two concentrations of dexamethasone, known to inhibit ARH77 cell growth. The growth of the control cells was markedly suppressed at 10−5 and 10−6M dexamethasone concentrations compared to cells without dexamethasone. However, p53as-transduced cells showed growth at these concentrations similar to cells without dexamethasone over period of 6 days of observation (Fig. 7).

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Fig. 7. Effect of dexamethasone on cell growth. Control and p53as-transduced ARH77 cells were exposed to dexamethasone at 10−5 and 10−6M concentrations for 6 days and number of trypan blue negative cells were measured on days 2, 4 and 6. Cell survivals were also measured with out dexamethasone treatment. p53as-transduced cells show continued growth compared to marked inhibition of the growth in control cells at both the concentrations.

4. Discussion 

A variety of cytogenetic and molecular alterations have been proposed to describe the evolution of myeloma from a benign monoclonal gammopathy of unknown significance to aggressive myeloma. Altered regulation in the IL-6/ras pathway, changes in bcl-2 and myc expression, and dysregulated retinoblastoma and p53 genes have been implicated in the progression of the disease [20]. However, the sequence of events that lead to the malignant progression of the disease are not thoroughly understood. One of the commonly altered genes in many malignancies is p53 [21]. However, in myeloma, several observations suggest that alterations in the p53 gene may represent an important late event associated with progression to an aggressive form of the disease [7], [9], [22]. Neri et al. studied the frequency and type of p53 gene mutations in a series of 52 cases of multiple myeloma representing different clinical phases and forms of the disease. They analyzed p53 gene mutations in exons 5–9. Point mutations were detected in seven of 52 patients, and were associated with a more advanced and clinically aggressive acute/leukemic form of multiple myeloma (7 of 16–43%). Three of the patients with mutated p53 genes had been evaluated at an earlier clinical presentation when the disease was indolent and were found to be negative for p53 gene mutations.

The role of altered p53 in myeloma genesis remains speculative. The major function attributed to p53 is the control of events leading to programmed cell death [11], [23]. p53 has been demonstrated, in a murine model, to induce differentiation of pre-B-cells [24]. In this study, we evaluated the role of p53 in myeloma cell growth by antisense RNA methodology. Although ARH77 cells are known to have normal p53, the antisense construct is likely to inhibit both wild-type and the mutated forms of p53. The observed increased cell growth, after the suppression of p53 expression, could be expected because of the known tumor suppressor function of p53. Our results suggest that up-regulation of bcl-2 expression may represent a mechanism to control programmed cell death in these cells. By suppressing p53 levels in our cell line, the concomitant bcl-2 up-regulation would result in decreased spontaneous apoptosis, thus leading to greater cell growth. Recent reports support such a mechanism. Over expression of the p53 gene has been shown to down-regulate bcl-2 expression, at both the protein and mRNA levels [25]. Other observations confirm that increased expression of p53, controlled by temperature-sensitive p53 induction, suppresses bcl-2 gene expression in variety of cell line models [26]. In addition to reporting p53 regulation of the bcl-2 gene in vitro, Miyshita et al. have shown an increase in bcl-2 in p53-deficient mice, as determined by both immunohistochemical and immunoblot methods. Further studies have shown a 195 bp segment in the bcl-2 gene 5′ untranslated region to be capable of conferring p53 dependent repression in heterologous expression plasmid system [27]. This provides a mechanism whereby p53 negatively controls bcl-2 transcription and programmed cell death. Our observation of down-regulation of p53 expression leading to increased bcl-2 expression is consistent with this information.

Multiple myeloma is a chemo sensitive disease in which more than 70% of the patients respond to standard-dose chemotherapy and more than 90% respond to high-dose chemotherapy. Dexamethasone, a steroid derivative, is a potent inducer of apoptosis in myeloma cells and remains an important agent for treatment. However, the natural history of myeloma suggests a gradual development of resistance to dexamethasone and chemotherapeutic agents leading to unchecked progression of the disease. Increasingly, the role of bcl-2 is being studied in relation to drug resistance [28], [29], [30]. Cell lines transduced with bcl-2 have been shown to develop resistance to chemotherapeutic and DNA-damaging agents [28]. Our current study shows that increased expression of bcl-2 or decreased p53 activity, independently or together, may be related to decreased apoptosis, providing dexamethasone resistance. It is conceivable that p53, predominantly involved in aggressive and terminal phases of myeloma, may lead to resistant disease with changes in bcl-2 expression and eventually a fatal outcome. Such a situation, if confirmed in primary tumor cells, may provide us with insight into various therapeutic options that can overcome increased bcl-2 expression and allow effective chemotherapeutic options.

The role bcl-2 plays in myeloma progression need further investigation. Our study suggests that it may play a significant role at various levels. It may be important for increased survival of the cells leading to larger tumor burden; it may be associated with p53 and other changes at a molecular level, that are associated with poor prognosis; and it may be responsible for chemotherapy and steroid therapy resistance leading to eventual uncontrolled tumor growth. In summary, our data demonstrates that loss of p53 function leads to myeloma cell progression and resistant phenotype through bcl-2-related mechanisms.