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Keywords:

  • apoptosis;
  • bcl-2;
  • prostatic neoplasms

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

AbstractBackground: Cancer cells often develop mechanisms to resist apoptosis and the extent of such anti-apoptotic ability has been shown to parallel tumor progression in various malignancies. Among various molecules implicated in regulating apoptosis pathway, bcl-2 and its family members are best characterized.

Methods: To investigate the effect of bcl-2-mediated anti-apoptotic ability on tumor growth and progression in prostate cancer, a cell line overexpressing bcl-2 (LNCaP/bcl-2) was established by genetically engineering a prostate cancer cell line LNCaP. Tumor growth of LNCaP/bcl-2 was compared with the parental cell line in vitro and in vivo.

Results: LNCaP/bcl-2 cells show resistance to apoptosis caused by nutrient deprivation and did not arrest when cultured in serum-free or androgen-free medium, while parental LNCaP cells or LNCaP cells transfected with the vector only (LNCaP/control) underwent extensive apoptosis on nutrient deprivation and sustained growth suppression in serum-free or androgen-free medium. When injected subcutaneously into nude mice, tumors deriving from LNCaP/bcl-2 cells grew faster compared with LNCaP/control for about 3 weeks (P = 0.02), but this effect was not evident after 5 weeks. Upon castration, the control tumors regressed but LNCaP/bcl-2-derived tumors showed resistance, as was previously reported.

Conclusions: These data confirm the notion that anti-apoptotic function of bcl-2 is oncogenic and confers resistance to androgen deprivation and also indicate that it may also play a critical role in earlier stages of tumorigenesis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Prostate cancers generally show androgen-dependent growth and undergo regression in response to androgen ablation by castration. 1,2 However, this treatment generally remains palliative and most eventually recur 3 and present androgen-independent growth capacity as well as resistance to other treatment modalities, like chemotherapy. 4–6 It is now widely accepted that apoptosis, a genetically programmed process of autonomous cell death, is the mechanism of this regression and recurrent tumors develop anti-apoptotic mechanism.

Apotosis is regulated by certain proto-ongogenes and the p53 tumor suppressor gene. The c-myc expression has been shown to be involved in the initiation of apoptosis in some situations. In the normal rat ventral prostate, castration results in regression of prostate volume by apoptosis and there is enhanced expression of gene for c-myc and c-fos. 7 In p53-negative tumor-derived cell lines transfected with wild-type p53, induction of the gene has been found to cause extensive apoptosis. However, p53 knock-out mice have apoptosis in the prostate glands after castration. 8 From the analysis of clinical specimens, p53 gene inactivation is rare in primary prostatic tumors not essential to the development of prostate cancer metastases. 9

Among various molecules identified as positive and negative regulators of apoptosis, bcl-2 is suggested to be a key molecule associated with anti-apoptotic capacity of prostate cancers, 10,11 as it is indeed upregulated in hormone refractory prostate cancers. 12,13 However, apoptosis can be induced by many other factors like irradiation, chemotherapy and nutrient deprivation, 14,15 and malignant potential in general seems to be correlated with anti-apoptotic capacity. To investigate the actual effect of anti-apoptotic capacity on prostate cancer growth and progression, we utilized a genetically engineered prostate cancer cell line which overexpresses bcl-2 and compared the tumor growth in vitro and in vivo with the parental cell line.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Cell culture and in vitro growth

An androgen-sensitive cell line LNCaP was purchased from American Type Culture Collection (Rockville, MD, USA). Cells were maintained at 37 °C in a humidified incubator with 5% CO2 and maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics. When the cells reached 50% confluence, the medium was changed to RPMI 1640 with 10% dextran-coated charcoal (DCC) treated FBS for androgen deprivation, RPMI1640 for serum deprivation, or Ca2+/Mg2+-free phosphate buffered saline (PBS) for nutrient deprivation. The cells were incubated for another 72 h and counted. Relative growth was calculated as (cell count after 72 h)/(cell count at the beginning of incubation).

Bcl-2 transfection in LNCaP cells

The bcl-2 transfectant of LNCaP cells (LNCaP/bcl-2) was made, using a bcl-2 expression vector pBC140 16 and Lipofectin Kit (Life Technology, Grand Island, NY, USA). A transfectant with a vector containing lacZ was also isolated (LNCaP/control). The bcl-2 protein expression of individual clone was examined by western blot analysis following a standard protocol.

DNA fragmentation assay

The DNA fragmentation assay was performed as previously described. 17 Briefly, the cells were lyzed in 100 μL TE9S buffer (500 mmol/L Tris-HCl, 2 mmol/L EDTA, 10 mmol/L NaCl, 1% [w/v] SDS) containing 1 mg/mL proteinase K and were incubated at 48°C for 24 h. DNA was extracted by phenol/chloroform extraction and was treated with RNase for 24 h at 37 °C. DNA obtained from 1 × 106 cells was run on a 1% agarose gel and visualized by ethidium bromide staining.

In vivo tumor growth

Male BALB/c nude mice were purchased from Nippon Bio-Supp. Center (Tokyo, Japan). Parental LNCaP cells, LNCaP/control cells or LNCaP/bcl-2 cells were suspended at a concentration of 2 × 10 6 cells/mL in RPMI 1640/10% FBS. This was then mixed with an equal volume of Matrigel basement membrane matrix (Becton Dickinson Labware, NJ, USA), 0.5 mL aliquots (0.5 × 10 6 cells) were injected subcutaneously into the flank of a mouse. Tumor growth was monitored by daily measurements of length (l), width (w) and depth (d). Tumor volume (V) was calculated by the formula: V = 0.5236 × l × w × d. After 5 weeks, the mice were castrated and were further monitored for the tumor size.

For statistical analysis of the reduction of tumor volume after castration, the two-tailed paired t-test was used and for the others, the unpaired t-test was used.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Establishment of bcl-2 overexpressing LNCaP cells

Western blot analysis revealed the parental LNCaP cells and LNCaP/control cells expressed only a marginal level of bcl-2 protein, while two transfectants, LNCaP/bcl-2-1 and LNCaP/bcl-2-2, showed high levels of bcl-2 expression ( Fig. 1). LNCaP/bcl-2–2 was chosen for further experiments as it showed stronger expression.

image

Figure 1. Western blot analysis of the expression of bcl-2 protein. LNCaP, parental LNCaP cell line; LNCaP/ control, control LNCaP transfected with vector alone; LNCaP/bcl-2-clone 1 and -clone 2, bcl-2 transfected LNCaP clones. The bcl-2 protein has a molecular weight of approximately 26 KDa.

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Overexpression of bcl-2 suppress apoptosi in vitro

Androgen deprivation by DCC caused significant growth suppression in parental LNCaP cells and LNCaP/control cells, with growth rates of 1.71 and 1.69, respectively, compared with the rates of 2.22 and 2.04 in FBS (P = 0.016), but did not have any effect on LNCaP/bcl-2 cells. Upon serum deprivation, further suppression was observed in LNCaP cells and LNCaP/control cells, with the growth rate 1.06 and 1.03 respectively. LNCaP/bcl-2 cells also had slight growth suppression, with the growth rate 2.34–2.05, but only 12.4% less than in FBS, showing serum- independent growth capacity ( Fig. 2). Under phase contrast microscopy, apoptotic cells were not evident in any cells, in either condition. When the cells were maintained in nutrient-free PBS, however, LNCaP/ control cells gradually changed their shape from a flat epitheloid appearance to a round shrunken one accompanied by several small and round bleb-like structures; namely, they underwent apoptosis. Morphologically apoptotic cells reached 20.0% of total cells at 48 h and 45.3% at 72 h. LNCaP/bcl-2 cells showed resistance to nutrient-free medium induced apoptosis, with only 8.4% apoptotic cells at 48 h and 17.0% at 72 h (data not shown). DNA extracted at each step showed characteristic DNA fragmentation pattern, confirming the morphologic observation ( Fig. 3).

image

Figure 2. The relative amount of cell growth in the test medium. Columns represent the mean; the bar represents the SD; n = 4 for each group. *P = 0.016, **P < 0.01 compared with in fetal bovine serum. FBS, Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum; DCCFBS, RPMI 1640 medium with 0.05% dextran-coated 0.5% charcoal-treated 10% fetal bovine serum; RPMI, RPMI 1640 medium.

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image

Figure 3. Electrophoresis of DNA extracted from LNCaP/control (LNCaP transfected with vector alone) and LNCaP/bcl-2 (LNCaP transfected with bcl-2) cells cultured in phosphate buffered saline for 2 days. The DNA of each cell was extracted and electrophoresis were carried out. On day 0, DNA fragmentation could not be observed in any cells. On day 1, a ladder pattern of discontinuous DNA bands could be barely detected in LNCaP/control cells and on day 2, the ladder became clear. However, no lane showed a ladder pattern in LNCaP/bcl-2. bp, Base pairs.

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In vivo tumorigenesis

In the pilot study, seven out of eight mice inoculated with LNCaP/bcl-2 cells formed macroscopic tumors, but only four of eight with LNCaP/control cells formed tumors. In following experiments, all eight mice with established macroscopic tumors of LNCaP/control and LNCaP/bcl-2 were used. The growth rate in the cases showing tumor formation was followed to record the in vivo growth effect of bcl-2 expression. For the first 21 days, LNCaP/bcl-2-derived tumors grew significantly faster than LNCaP/ control-derived tumors. The average volumes on the day 21 were 75.7 ± 27.5 mm3 for LNCaP/bcl-2 and 8.0 ± 5.4 mm3 for LNCaP/control (P = 0.02). By day 35, however, the size of the tumors in all the mice had reached similar sizes of 101.8 ± 34.8 mm3 and 128.5 ± 14.7 mm, 3 for LNCaP/bcl-2 and LNCaP/control, respectively ( Fig. 4). Following the castration, all the tumors diminished in size and LNCaP/control tumor volume was significantly reduced (P < 0.01). The reduction rate 18 days following castration was 8.5% for LNCaP/bcl-2 and 71.9% for LNCaP/control derived tumors.

image

Figure 4. Growth of LNCaP/control tumor and LNCaP/bcl-2 tumor in nude mice. The castration was performed on day 35. (○), the mean volume of LNCaP/control tumors (n = 8); (●), LNCaP/bcl-2 (n = 8); bar, SE. *P = 0.02 between LNCaP/ control and LNCaP/bcl-2 at day 21; **P < 0.01 compared with the LNCaP/control tumor volume on day 35.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

In this study, we first established a cell line which stably expresses bcl-2 and confirmed in vitro that exogenous expression of bcl-2 confers resistance to apoptosis induced by nutrient deprivation. On serum or androgen deprivation, however, the cells underwent growth suppression but not apoptosis. Even so, the overexpression of bcl-2 did have an effect under such conditions and overcame the growth suppression. A previous study using the same cell line and the same strategy reported that apoptosis was induced on serum deprivation, 18 which was not obvious in our study. There are two possible explanations for the effect of bcl-2 in in vitro growth. At first there may have been apoptosis on serum/androgen deprivation which was not detectable by our method and which was suppressed by bcl-2 expression resulting in an increase of total cell growth. Alternatively, bcl-2 overexpression may have blocked the growth suppression caused by serum/androgen deprivation by a mechanism other than anti-apoptosis. Current knowledge on bcl-2 function strongly supports the former explanation, 19 but the latter cannot be excluded completely.

The effect of bcl-2 overexpression on in vivo tumorigenesis may better be considered in three separate phases, as each may have different clinical significance: (i) establishment of a solid tumor capable of continuous growth; (ii) growth rate of the tumor; and (iii) response to the castration. Interestingly, our data presented here indicate bcl-2 exhibits its potential as a proto-oncogene in all three phases. First, although the number was too small to reach statistical significance, the bcl-2 transfectant formed solid tumors easier than controls. The rate of LNCaP/bcl-2 tumor formation was 87.5% (7/8). In contrast, the rate of control tumor formation was 50.0% (4/8). Inoculated cells are under stress by an unfavorable micro- environment, which presumably stimulates the apoptotic pathway in the cells; bcl-2 would help to overcome such apoptotic stimuli and increase the likelihood of establishing a solid, growing tumor. In support of this, a recent study analyzing metastasis-prone subclones obtained from LNCaP cells showed metastatic potential was accompanied by bcl-2 expression. 20 Second, bcl-2 conferred faster growth once the tumor was established. This is in accordance with our results in vivo. The established macroscopic tumors of LNCaP/bcl-2 grew significantly faster than LNCaP/control derived tumors during the first 21 days. A similar explanation is possible. The tumor formation is now understood to be the result of an inbalance between cell proliferation and cell death. 21 If cell proliferation and cell death are thought of as a ratio, then tumor mass will increase if proliferation increases or death decreases (proliferation/death > 1). In the early stages of tumor growth, the number of tumor cells are low and some stress by an unfavorable micro- environment, like an immune reaction of the host or a shortage of autocrine or paracrine growth factors, may cause the tumor cells to undergo apoptosis. It should be noted that the growth advantage was not obvious after 35 days when the tumor mass reached around 120 mm3 in size. One possible explanation is that the tumor size beyond a certain level may be limited by factors which are not affected by bcl-2-mediated anti-apoptotic capacity. For instance, shortage of nutrition could limit the cell growth by mechanisms which are not influenced by bcl-2. In that sense, it would be interesting to see the effects of the development of new blood vessels 22–25 using angiogenetic factors such as bFGF or VEGF 26 in a similar model and compare these results with the present study. Third, as previously described by another group, 18 bcl-2 overexpression bestowed resistance to androgen ablation. These effects of bcl-2 in the three phases would correspond to three characteristic features of malignant tumors; metastatic ability, faster growth and resistance to therapy. Our data thus demonstrated that bcl-2 confers features of high grade tumors and underscores the significance of bcl-2 overexpression frequently observed in recurrent prostate cancers.

Various molecules are involved in the control of the apoptosis pathway and bcl-2 is only one such molecule. However, our study using a simple model illuminated the importance of anti-apoptotic capacity in the recurrence and progression of prostate cancer. Further improvement of prostate cancer treatment may rely on measures which could alter the features of progressed cancers, which, at least in part, are mediated by anti-apoptotic capacity.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We thank Ms E Tanaka for her assistance in cell culture and Dr Y Tsujimoto for the kind gift of the bcl-2 expression vector pBC140.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • 1
    Huggins C, Stevens RE, Hodges CL. Studies on prostatic cancer. II. The effect of castration on clinical patients with carcinoma of the prostate. Arch. Surg. 1941; 43: 209.
  • 2
    Huggins C & Hodges CV. Studies on prostatic cancer; effect of castration, of estrogen and of androgen injection on serum phosphatases in metastatic carcinoma of the prostate. Cancer Res. 1941; 1: 293 7.
  • 3
    Sinha AA, Blackard CE, Seal US. A critical analysis of tumor morphology and hormone treatments in the untreated and estrogen-treated responsive and refractory human prostatic carcinoma. Cancer 1977; 40: 2836 50.
  • 4
    Emmett JL, Greene LF, Papantoniou A. Endocrine therapy in carcinoma of the prostate gland: 10-year survival studies. J. Urol. 1960; 83: 471 84.
  • 5
    Raghavan D. Non-hormone chemotherapy for prostate cancer: Principles of treatment and application to the testing of new drugs. Semin. Oncol. 1988; 15: 371 89.
  • 6
    Kres W. Current chemotherapy and future directions in research for the treatment of advanced hormone- refractory prostate cancer. Cancer Invest. 1995; 13: 296 312.
  • 7
    Buttyan R, Zaker Z, Lockshin R, Wolgemuth D. Cascade induction of c-fos, c-myc and heat shock 70K transcripts during regression of the rat vetral prostate gland. Mol. Endcrinol. 1988; 2: 650 7.
  • 8
    Colombel M, Radvanyi F, Blanche M et al. Androgen suppressed apoptosis is modified in p53 deficient mice. Oncogene 1995; 10: 1269 74.
  • 9
    Brooks JD, Bova GS, Ewing CM et al. An uncertain role for p53 gene alterations in human prostate cancers. Cancer Res. 1996; 56: 3814 22.
  • 10
    Sinha BK, Yamazaki H, Eliot HM, Schneider E, Borner MM, O’Connor PM. Relationships between proto-oncogene expression and apoptosis induced by anticancer drugs in human prostate tumor cells. Biochem. Biophys. Acta 1995; 1270: 12 18.
  • 11
    Berchem. GJ, Bosseler M, Sugars LVY, Voeller HJ, Zeitlin S, Gelmann EP. Androgens induce resistance to bcl-2-mediated apoptosis in LNCaP prostate cancer cells. Cancer Res. 1995; 55: 735 8.
  • 12
    McDonnell TJ, Troncoso P, Brisbay SM et al. Expression of the protooncogene bcl-2 in the prostate and its association with emergence of androgen-independent prostate cancer. Cancer Res. 1992; 52: 6940 4.
  • 13
    Colombel M, Symmans F, Gil S et al. Detection of the apoptosis-suppressing oncoprotein bcl-2 in hormone-refractory human prostate cancer. Am. J. Pathol. 1993; 143: 390 400.
  • 14
    Arends MJ, Morris RG, Wyllie AH. Apoptosis: The role of the endonuclease. Am. J. Pathol. 1990; 136: 593 608.
  • 15
    Wyllie AH. Apoptosis (The 1992 Frank Rose Memorial lecture). Br. J. Cancer 1993; 67: 205–8.
  • 16
    Borzillo GV, Endo K, Tsujimoto Y. Bcl-2 confers growth and survival advantage to interleukin 7- dependent early pre-B cells which become factor independent by a multistep process in culture. Oncogene 1992; 7: 869 76.
  • 17
    Kauffmann SH. Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, campthecin, and other cytotoxic anticancer drugs: A cautionary note. Cancer Res. 1989; 49: 5870 8.
  • 18
    Raffo AJ, Perlman H, Chen M-W, Day ML, Streitmean JS, Buttyan R. Overexpression of bcl-2 protects prostate cancer cells from apoptosis in vitro and confers resistance to androgen depletion in vivo. Cancer Res. 1995; 55: 4438 45.
  • 19
    Kerr JFR, Winterford CM, Harmon BV. Apoptosis: Its significance in cancer and cancer therapy. Cancer 1994; 73: 2013 26.
  • 20
    McConkey DJ, Green G, Pettaway CA. Apoptosis resistance increases with metastatic potential in cells of the human LNCaP prostate carcinoma line. Cancer Res. 1996; 56: 5594 9.
  • 21
    Manning FCR & Patierno SR. Apoptosis: Inhibitor or instigator of carcinogenesis? Cancer Invest. 1996; 14: 455 65.
  • 22
    Weidner N, Carrol PR, Flax J, Blumenfeld W, Folkman J. Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am. J. Pathol. 1993; 143: 401 3.
  • 23
    Bigler SA, Brawer MK, Deering RE. Comparison of microscopic vascularity in benign and malignant prostatic tissue. Human Pathol. 1993; 24: 220 6.
  • 24
    Wakui S, Furusato M, Itoh T et al. Tumor angiogenesis in prostatic carcinoma with and without bone marrow metastasis: A morphometric study. J. Pathol. 1992; 168: 257 62.
  • 25
    Furusato M, Wakui S, Sasaki H, Itoh T, Ushigome S. Tumor angiogenesis in latent prostatic carcinoma. Br. J. Cancer 1994; 70: 1244 6.
  • 26
    Polverini PJ. Macrophage induced angiogenesis: A review. Cytokines 1989; 1: 54 73.