I. Introduction
Benign
enlargement of the prostate, or benign prostate hyperplasia (BPH), is the most
commonly occurring benign disease in aging men (Walsh, 1984). The incidence of
BPH increases with advancing age, so that 50% of all men older than 50 years of
age have symptoms arising from BPH (Walsh, 1984; McConnell, 1990). In over 25%
of men over the age of 60, surgery is necessary (Walsh, 1984). Although these
epidemiological facts underscore the importance of BPH as a major health
problem, there is no specific or effective therapeutic modalities selectively
targeted for BPH currently available (McConnell, 1990). Cell growth in the
normal prostate is regulated by a delicate balance between cell death and cell
proliferation. Disruption of the molecular mechanisms that regulate these two
processes may underline the abnormal growth of the prostate gland leading to
BPH (Kyprianou et al, 1996). Several genes which serve as growth negative
regulators, or so-called gate keepers such as p16, p53, and TGFb1,
control cellular proliferation by either cell cycle arrest or induction of
programmed cell death (apoptosis), and therefore, maintain the critical balance
between cell proliferation and cell death in normal prostate homeostasis.
The p16 gene product, also
called MTS1, INK4A, and CDKN2, is a cyclin dependent kinase inhibitor and an
important negative cell cycle regulator whose functional loss may significantly
contribute to malignant transformation (Serrano et al, 1993). Inactivation of
p16 tumor suppressor gene is one of the most common events in a wide range of
tumors including malignant melanoma (Talve et al, 1997), mesothelioma (Xiao et
al, 1995), leukemia (Shapiro and Rollins, 1996), glioma, carcinomas of the
lung, colon, liver, kidneys, esophagus, pancreas, breast, ovary, biliary tract
and prostate, and sarcomas (Okamoto et al, 1994; Cairns et al, 1995; Liu et al,
1995; Smith-Sorenson and Hovig, 1995; Talve et al, 1997). These
alterations in p16 may be a result of DNA mutations (Caldas et al, 1994; Kamb
et al, 1994; Mori et al, 1994; Nobori et al, 1994), homozygous loss of p16 gene
at the DNA level (Jen et al, 1994), or methylation of the promoter region at
epigenetic level (Smith-Sorenson and Hovig, 1995). Restoration of p16
expression has resulted in suppressed growth of a variety of tumor types
including human prostate cancer cells (Gotoh et al, 1997), nonsmall cell
carcinoma of the lung (Jin et al, 1995), glioma cells (Arap et al, 1995),
prostate carcinoma (Gotoh et al, 1997) and hepatocellular carcinoma (Sandig et
al, 1997), and others (Serrano et al, 1995; Quesnel et al, 1996; Schrump et al,
1996; Stone et al, 1996; Sandig et al, 1997). p53 is a nuclear phosphoprotein
that acts as a transcription factor to control the expression of proteins
involved in the cell cycle (Selter, 1994; Ozbun and Butel, 1995). In response
to DNA damage, p53 accumulates in the nucleus and can arrest the cell cycle via
the cyclin-dependent kinase inhibitor p21/Waf1. However, in neoplastic cells
p53 usually induces apoptosis, or programmed cell death. Mutations in p53 have
been reported in a majority of malignant cell types, and it has been estimated
that p53 function is altered in half of all human malignancies (Nielsen and
Maneval, 1998). Introduction of wild type p53 has been shown to inhibit the
tumorigenic phenotype of many tumor cell lines (Nielsen and Maneval, 1998). TGFb1,
one of the most potent physiological negative regulators of epithelial cell
growth, exerts its effect by binding to a cell surface receptor and triggering
a signaling pathway that leads to the modulation of factors that directly
regulate the cell cycle such as c-Myc, cyclins and cyclin-dependent kinases
(Alexandrow and Mose, 1995). TGFb1
in general stimulates mesenchyme and inhibits epithelial growth (Moses et al,
1990). Addition of TGFb1
to cultured prostatic epithelial and stromal cells has been reported to inhibit
proliferation in both cell types (Sutkowski et al, 1992). It is suggested that
TGFb1 may
exert its growth inhibition via upregulation of other growth negative regulator
genes such as p15 (Hannon and Beach, 1994) and p21 (Macleod et al, 1995). In
this study, we examined whether the exogenous expression of the negative growth
regulators p16, p53 and TGFb1
will lead to growth inhibition of the BPH prostate.
II.
Materials and methods
A. Generation of recombinant
adenovirus Adp16, Adp53, and AdTGFb1
Details of
the construction of adenovirus expressing foreign cDNA gene have been
previously described (Steiner et al, 2000). For construction of the adenovirus
expressing wild type p16 (Adp16), the 960-bp human p16 cDNA gene was placed in
an E1-deleted adenoviral shuttle vector under the control of the Rous sarcoma
virus (RSV) promoter. The resultant adenoviral shuttle vector was cotransfected
into 293 cells with pJM17, an adenoviral genome plasmid, by the calcium
phosphate method (Kingston, 1993). The individual plaques were screened by
direct plaque screening PCR method (Lu et al, 1998) using specific primers for
the RSV promoter and for p16 the cDNA gene to identify the Adp16 plaque. The
resultant Adp16 is a replication defective, recombinant adenoviral vector.
Similarly, adenovirus expressing TGFb1 (AdTGFb1) was generated, in which TGFb1 gene was modified so the resultant protein product TGFb1 is an active form of TGFb1 (Pierce et al,
1999). Adenovirus expressing wild type p53 gene under the control of the
cytomegalovirus (CMV) promoter was a gift from University of Texas MD Anderson
Cancer Center.
B. Adenovirus preparation,
titration and transduction
Individual
clones of Adp16, Adp53, and AdTGFb1
were obtained by three-time plaque purification. Single viral clones were then
propagated in 293 cells. The culture medium of the 293 cells showing a complete
cytopathic effect was collected and adenovirus was purified and concentrated by
twice CsCl2 gradient ultracentrifugation. The viral titration was performed as
described (Graham and Prevec, 1991). Viral transduction was performed at
various multiplicity of infection (moi) in a volume of 1 ml culture medium and
incubated at 370C with gentle mixing every 15 min. After a 90-min
incubation, the infectious supernatant was then replaced with fresh medium.
C. Cell culture and medium
DulbeccoÕs modified Eagle
medium (D-MEM) and RPM1-1640 were purchased from Gibco BRL (Gaithersburg, MD).
Fetal bovine serum (FBS) was from Hyclone Laboratories (Logan, UT). Human
embryonic kidney 293 cells were grown in D-MEM with 10% heat inactivated FBS.
The BPH-1 cell line, an immortalized but non-transformed human prostate-derived
epithelial cell line (Hayward et al, 1995), was grown in RPMI-1640 medium with
10% FBS. All cell lines were grown in medium containing 100 units/ml
penicillin, 100 mg/ml streptomycin at 370C
in 5% CO2.
D. Northern blot analysis
Cells were
extracted and total RNA was isolated by RNeasy Total RNA Kit (Qiagen, Santa
Clarita, CA). Total RNA (10 mg) was loaded on a
1.2% polyacrylamide gel and electrophoresed. The standard Northern blot
transfer to a Nylon membrane (Hybond-N+,
Amersham Life Science, Buckinghamshire, England) was performed as previously
described (Sambrook et al, 1989). The cDNA probe (p16, p53 and TGFb1, respectively) was labeled by a-32P-dCTP
using random primer method (Prime-It II Kit, Stratagene, La Jolla, CA). The
membrane was hybridized with the probe in Rapid-hyb buffer (Amersham Life
Science) according to the manufacturer's protocol. The membrane was exposed to
a Kodak X-ray film between two intensifying screens at -800C for
autoradiography. The cDNA probe for glyceradehyde 3-phosphate dehydrogenase
(GAPDH) was labeled as described above and used as an internal control for
normalization.
E. ELISA assay
Cells were grown and maintained in the medium containing
10% charcoal-stripped FBS for this experiment. Supernatants were collected at
24 h, 48 h, 72 h, respectively, after viral transduction. The immunoassay for
TGFb1 production was determined by a Quantikine human TGFb1 ELISA kit (R&D Systems Inc., Minneapolis, MN) according to the
manufacturer's manual.
F. Growth inhibition assay
Cells were plated at 5x103
cells per well in 6-well plates; the next day the cells were incubated at 370C
untreated or transduced by adenovirus. The cell numbers were counted in
triplicate by a Coulter cell counter six days after transduction.
G. DNA
extraction and gel electrophoretic analysis of DNA fragmentation
Soluble DNA was extracted as described previously (Oridate et al,
1996). Briefly, the cells floating in medium were collected at 48 h, 72 h and
96 h post transduction by centrifugation. The pellet was resuspended in
Tris-EDTA buffer (pH 8.0). The cells were lysed in 10 mM Tris-HCl (pH 8.0), 10
mM EDTA, and 0.5% Triton X-100 on ice for 15 min. The lysate was centrifuged at
12,000xg for 15 min to separate soluble (fragmented) DNA from pellet (intact
genomic) DNA. Soluble DNA was treated with RNase A (50 mg/ml) at 370C for 1 h,
followed by treatment with proteinase K (100 mg/ml) in 0.5% SDS, at 500C
for 2 h. The residual material was extracted with phenol/chloroform,
precipitated in ethanol, and electrophoresed on a 2% agarose gel.
H. TUNEL assay
Slide flasks
(NUNC, Roskilde, Denmark) were precoated with 50 mg/ml poly-L-lysine
(Sigma, St. Louis, MO) for 15 min and washed twice with PBS. BPH-1 cells (1x105)
were plated on slide flasks and grown for 24 h before viral transduction. The
cells were then either untreated or transduced with control virus AdlacZ or
Adp16, Adp53 and AdTGFb1, respectively,
at multiplicity of infection (moi) of 200. Cell monolayers grown on slides were
rinsed twice with PBS at 72 h after transduction and subjected to TUNEL
staining. In Situ Cell Death
Detection Kit, POD (Boehringer Mannheim Corp., Indianapolis, IN) was used for the TUNEL assay according
to the manufacturerÕs instruction and cells were visualized by fluorescence
microscopy.
I. Flow cytometry to
detect apoptotic population
Cells were
arrested at G2/M phase by treating cells with 400 ng/ml nocodazole (Sigma) at
370C for 12 to 16 h (Park and Koff, 1998). After washing the cells,
the synchronized cells were replated in fresh medium, and the cells were
untreated or treated with viral transduction. Cellular DNA content that
represents the cell cycle phase (G1, S, or G2/M) and apoptotic population (pre
G1) was detected by flow cytometry via determination of propidium iodide
staining (Lou and Xu, 1997; Zhang et al, 1999). After trypsinization, cells
were washed with PBS and cell pellets were fixed in 70% ethanol at 40C
overnight. After two washes with PBS, the cells were stained with 50 mg/ml propidium iodide (Sigma, St. Louis, MO), 10 mg/ml RNase A, 0.5% (v/v) Triton X-100 and 0.1% (w/v) sodium citrate
for 30 min at room temperature in the dark followed by fluorescent flow
cytometry.
III. Results
A. Adenoviral transduction
resulted in overexpression of p16, p53 and TGFb1 in BPH-1 cells
To determine whether the adenoviral vector served as a
gene transfer vehicle that transduced BPH cells, an adenovirus carrying an E.
coli lacZ reporter gene with a nuclear localization signal at the 5Õ upstream
(AdlacZ) (previously (Oridate et al, 1996).) was used to transduce BPH-1. At a
moi of 200, AdlacZ resulted in greater than a 95% transduction rate in BPH-1
cells as indicated by the percentage of cells that exhibited blue staining (not
shown). To determine whether the adenovirus successfully transferred and
expressed p16, p53 or TGFb1, BPH-1 cells were
transduced by adenovirus expressing p16 (Adp16) or TGFb1 (AdTGFb1) under the control of RSV promoter, or by adenovirus expressing p53
(Adp53) under the control of CMV promoter at moi of 200, respectively. The cell
extracts were harvested 48 h after viral transduction and the mRNA was
isolated. Under our experimental conditions, Northern blot analysis showed that
there was no detectable endogenous p16, p53, and TGFb1 in BPH-1 cells; however, there was a high exogenous
mRNA expression of p16, p53 and TGFb1, at 0.9 kb, 2.5 kb, and 2.5 kb transcript size, respectively, in the
cells transduced by each corresponding adenovirus (Figure 1). In addition, culture media of BPH-1 control cells, cells
transduced by AdlacZ control virus and AdTGFb1 were collected at specific time points and analyzed for secreted TGFb1 expression by an ELISA assay. As illustrated by Table 1, there was an increased TGFb1 protein in the medium from AdTGFb1 transduced BPH-1 cells that correlated with moi, except for the 72 h
point with a moi of 500, which had an decreased TGFb1 expression. This latter result was probably due to
the direct cytoxicity of the adenovirus at this high moi which decreased cell
number. In contrast, untreated BPH-1 cells and cells transduced by control
virus did not show detectable TGFb1 secretion. These
al, 1996), breast cancer (Nielsen et al, 1997; Xu et
al, 1997), cervical cancer (Hamada et al, 1996), ovarian carcinoma (Mujoo et
al, 1996), and melanoma (Cirielli et al, 1995). A retroviral vector expressing
BRCA1 tumor suppressor gene was used in Phase I human clinical trial for
advanced prostate cancer (Steiner et al, 1998). Adenovirus expressing p16 gene
significantly inhibited prostate cancer growth and prolonged survival in an
animal model (Steiner et al, 2000). Therefore, it is possible that adenoviral
vectors expressing p16, p53, and TGFb1 may have a therapeutic potential for BPH therapy.
To
determine whether overexpression of these negative growth regulators would lead
to cell growth inhibition, recombinant adenoviruses expressing p16, p53, and
TGFb1 were used to transduce an established human BPH cell
line, BPH-1, and the potential inhibitory effect was evaluated. The expression
of exogenous p16, p53, and TGFb1
via adenoviral-mediated gene transfer inhibited cell growth and induced
apoptosis in BPH-1 cells. Interestingly, while p16 showed the strongest
inhibitory effect on BPH-1 cell growth among the three negative growth
regulators (Figure 2), it did not
appear to lead to the highest apoptotic population (Figure 5 and Table 2). One explanation is that p16 may have dual
inhibitory mechanisms in BPH-1 cells, composed of apoptosis and other
inhibitory mechanisms such as cell cycle arrest. As p16 has been reported
previously to be able to induce senescence (Kingston, 1993; Steiner et al,
2000), the observed growth inhibition by Adp16 in BPH-1 cells may be a result
of the combination of apoptosis and senescence. Moreover, our
recent studies showed that p16 inhibits VEGF expression and suppresses
neovascularization and angiogenesis in breast cancer cells (Lu et al., Cancer Therapy, 1: 143-151,
2003) and prostate cancer cells, as well as in BPH-1 cells (our unpublished
data). As VEGF and other angiogenic factors play a major role on cell
proliferation and survival, the anti-VEGF effect renders another level of p16Õs
anti-cell growth function. Thus, p16 can cause cell-cycle arrest, induce
apoptosis, senescence and anti-angiogenesis, which may account for it being as
the most potent cell-growth inhibitor (Figure
2), as cell growth result is a net result of the combination of all these
effects. While some reports showed that p53 is a more potent cell killer, the
seemingly less potent p53 cell-killing effect in our study may be cell specific
in this case. The BPH-1 cells was established by immortalizing human primary
prostate epithelial cells with SV40 large T antigen, BPH-1 line has an
increased endogenous levels of p53 (Hayward et al., 1995), which may
desensitize Adp53 effect in BPH-1 line as it already has a basal level of p53.
On the other hand, Adp53 clearly demonstrated a greater effect on apoptosis in
our study, as reflecting by flow cytometry data (Figure 5 and Table 2, see pre-G1/apoptosis cell population), which
is consistent with other reports.
The current therapies for BPH such as prostatectomy
are invasive with side effects. Furthermore, the current therapies cannot
arrest BPH progression. We hypothesize that molecular therapies may be less
toxic and invasive and potentially more effective. Overexpression of p16, p53
and TGFb1 leads to suppression of BPH cell growth and even
induction of apoptosis, suggesting that this approach may be useful as gene
therapy to reduce BPH and relieve urinary obstruction. Whether the combined use
of two or more of these adenoviruses on BPH cells will lead to a potential
synergistic inhibitory effect needs to be further examined. To our knowledge,
this is the first report to use a gene therapy approach evaluate its potential
to treat BPH disease. These studies support further studies using in vivo models such as BPH dogs followed
by prostate volume measurement as a treatment endpoint.
Acknowledgments
This research was supported in part by National
Institute of Health grant DK65962 (Y.L.), in part by Elsa U. Pardee Foundation
(Y.L.), and in part by Cancer Research and Prevention Foundation (Y.L.). We are
grateful to Dr. Simon Hayward (Vanderbilt
University) for the generous gift of BPH-1 cell line.
References
Alexandrow MG,
Mose, HL (1995) Transforming growth
factor b and cell cycle regulation. Cancer
Res 55, 1452-1457.
Arap W, Nishikawa
R, Furnari FB, Cavenee WK, Huang HJ (1995) Replacement of the p16/CDKN2 gene
suppresses human glioma cell growth. Cancer Res 55, 1351-1354.
Cairns P,
Polascik TJ, Eby Y, Sidransky D (1995)
High frequency of homozygous deletion at p16/CDKN2 in primary human tumors. Nat Genet 11, 210-212.
Caldas C, Hahn
SA, da Costa LT, Redston MS, Schutte M, Seymour AB, Weinstein CL, Hruban RH,
Yeo CJ, Kern SE (1994) Frequent
somatic mutations and homozygous deletions of the p16 Nat Genet 8, 27-32.
Cirielli C,
Riccioni T, Yang C, Pili R, Gloe T, Chang J, Inyaku K, Passaniti A, Capogrossi
MC (1995) Adenovirus-mediated gene
transfer of wild-type p53 results in melanoma cell apoptosis in vitro and in vivo. Int J Cancer 63,
673-679.
Gotoh A, Kao C,
Ko SC, Hamada K, Liu TJ, Chung LW (1997)
Cytotoxic effects of recombinant adenovirus p53 and cell cycle regulator genes J Urol
158, 636-641.
Graham FL, Prevec
L (1991) Gene transfer and
expression protocols. In: Methods in Molecular Biology. Murray EJ (ed). Vol.7.
Hamada K, Alemany
R, Zhang WW, Hittelman WN, Lotan R, Roth JA, Mitchell MF (1996) Adenovirus-mediated transfer of a wild-type p53 gene and induction
of apoptosis in cervical cancer. Cancer
Res 56, 3047-3054.
Hannon GJ, Beach
D (1994) p15 Nature 371, 257-261.
Hayward SW,
Dahiya R, Cunha GR, Bartek J, Deshpande N, Narayan P (1995) Establishment and characterization of an immortalized but
non-transformed human prostate epithelial cell line: BPH-1. In Vitro Cell Dev Biol 31, 14-24.
Herman JG, J, J,
Merlo A, Baylin SB (1996)
Hypermethylation-associated inactivation indicates a tumor suppressor role for
p15 INK4. Cancer Res 56,
722-727.
Jen J, Harper JW,
Bigner SH, Bigner DD, Papadopoulos N, Markowitz S, Willson JK, Kinzler KW,
Vogelstein B (1994) Deletion of p16
and p15 genes in brain tumors. Cancer
Res 54, 6353-6358.
Jin X, Nguyen D,
Zhang WW, Kyritsis AP, Roth JA (1995)
Cell cycle arrest and inhibition of tumor cell proliferation by the p16INK4
gene mediated by an adenovirus vector. Cancer
Res 55, 3250-3253.
Kamb A, Gruis NA,
Weaver-Feldhaus J, Liu Q, Harshman K, Tavtigian SV, Stockert E, Day RS 3rd,
Johnson BE, Skolnick MH (1994) A
cell cycle regulator potentially involved in genesis of many tumor types. Science 264, 436-440.
Kingston RE (1993) Calcium phosphate transfection.
In: Current Protocols in Molecular Biology. Ausubel FM, Brent R, Kingston RE et
al (eds). John Wiley & Sons, New York, pp 9.1.4-9.1.9.
Ko SC, Gotoh A,
Thalmann GN, Zhau HE, Johnston DA, Zhang WW, Kao C, Chung LW (1996) Molecular therapy with
recombinant p53 adenovirus in an androgen-independent, metastatic human
prostate cancer model. Hum Gene Ther 7,
1683-1691.
Kyprianou N, Tu
H, Jacobs SC (1996) Apoptotic versus
proliferative activities in human benign prostatic hyperplasia. Hum Pathol 27, 668-675.
Liu Q, Neuhausen
S, McClure M, Frye C, Weaver-Feldhaus J, Gruis NA, Eddington K,
Allalunis-Turner MJ, Skolnick MH, Fujimura FK, et al (1995) CDKN2 Oncogene 11,
2455.
Lou LG, Xu B (1997) a-Anordrin-induced
apoptosis of leukemia K562 cells is not prevented by cycloheximide. Acta Pharmacol Sin 18, 169-172.
Lu Y, Carraher J,
Zhang Y, Armstrong J, Lerner J, Rogers WP, Steiner MS (1999) Delivery of adenoviral vectors to the prostate for gene
therapy. Cancer Gene Ther 6, 64-72.
Lu Y, Zhang Y,
Steiner M (1998) Efficient
identification of recombinant adenoviruses by direct plaque-screening. DNA Cell Biol 17, 643-645.
Macleod KF, Sherry
N, Hannon G, Beach D, Tokino T, Kinzler K, Vogelstein B, Jacks T (1995) p53 dependent and independent
expression of p21 during cell growth, differentiation and DNA damage. Genes Develop 9, 935-944.
McConnell JD (1990) Medical management of BPH with androgen
suppression. Prostate 3, 49-59.
Mori T, Miura K,
Aoki T, Nishihira T, Mori S, Nakamura Y (1994)
Frequent somatic mutation of the MTS1/CDK4I Cancer Res 54, 3396-3397.
Moses HL, Yang
EY, Pietenpol JA (1990) TGFb1 stimulation and
inhibition of cell proliferation: new mechanistic insights. Cell 63, 245-247.
Mujoo K, Maneval
DC, Anderson SC, Gutterman JU (1996)
Adenoviral-mediated p53 tumor suppressor gene therapy of human ovarian
carcinoma. Oncogene 12, 1617-1623.
Nielsen LL, Dell
J, Maxwell E, Armstrong L, Maneval D, Catino JJ (1997) Efficacy of p53 adenovirus-mediated gene therapy against
human breast cancer xenografts. Cancer
Gene Ther 4, 129-138.
Nielsen LL,
Maneval D (1998) p53 tumor
suppressor gene therapy for cancer. Cancer
Gene Ther 5, 52-63.
Nobori T, Miura
K, Wu DJ, Lois A, Takabayashi K, Carson DA (1994) Deletions of the cyclin-dependent kinase-4 inhibitor gene in
multiple human cancers. Nature 368,
753-756.
Okamoto A,
Demetrick DJ, Spillare EA, Hagiwara K, Hussain SP, Bennett WP, Forrester K,
Gerwin B, Serrano M, Beach DH, Harris CC (1994) Mutations and altered expression
of p16INK4 in human cancer. Proc Nat Acad Sci USA 91, 11045-11049.
Oridate N, Lotan
D, Xu XC, Hong WK, Lotan R (1996)
Differentiation induction of apoptosis by all-trans-retinoic acid and
N-(4-hydroxyphenyl)retinamide in human head and neck squamous cell carcinoma
cell lines. Clin Cancer Res 2,
855-863.
Ozbun MA, Butel J
(1995) Tumor suppressor p53
mutations and breast cancer: a critical analysis. Adv Cancer Res 66, 71-141.
Park M, Koff A (1998) Methods for synchronizing cells
at specific stages of the cell cycle. In: Current Protocols in Cell Biology.
Bonifacino JS, Dasso M, Harford JB, Lippincott-Schwartz J and Yamada KM (eds).
John Wiley & Sons, Inc., New York, pp
8.3.1-8.3.16.
Pierce DF Jr,
Johnson MD, Matsui Y, Robinson SD, Gold LI, Purchio AF, Daniel CW, Hogan BL,
Moses HL (1999) Inhibition of
mammary duct development but not alveolar outgrowth during pregnancy in
transgenic mice expressing active TGFb1. Genes Dev 7, 2308-2317.
Quesnel B,
Preudhomme C, Lepelley P, Hetuin D, Vanrumbeke M, Bauters F, Velu T, Fenaux P (1996) Transfer of p16INKa/CDK2 gene in
leukaemic cell lines inhibits cell proliferation. Br J Haematol 95, 291-298.
Sambrook J,
Fritsch E, Maniatis T (1989)
Northern blot hybridization. In: Molecular Cloning. Sambrook J, Fritsch E,
Maniatis T (eds). Cold Spring Harbor Laboratory Press, Plainview, pp 7.43-7.50.
Sandig V, Brand
K, Herwig S, Lukas J, Bartek J, Strauss M (1997)
Adenovirally transferred p16INK4/CDKN2 and p53 genes cooperate to
induce apoptotic tumor cell death. Nature
Med 3, 313-319.
Schrump DS, Chen
GA, Consuli U, Jin X, Roth JA (1996)
Inhibition of esophageal cancer proliferation by adenovirally mediated delivery
of p16INK4. Cancer Gene Ther 3,
357-364.
Selter, H (1994) and Montenarh, M.: The emerging
picture of p53. Int J Biochem 26,
145
Serrano M,
Gomez-Lahoz E, DePinho RA, Beach D, Bar-Sagi D (1995) Inhibition of ras-induced proliferation and cellular
transformation by p16INK4. Science 267,
249-252.
Serrano M, Hannon
GJ, Beach D (1993) A new regulatory
motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 366, 704-707.
Shapiro GI,
Rollins BJ (1996) p16INK4A
as a human tumor suppressor. Biochem
Biophys Acta 1242, 165-169.
Smith-Sorenson B,
Hovig E (1995) CDKN2 Hum Mutation 7, 294-303.
Spitz FR, Nguyen
D, Skibber JM, Cusack J, Roth JA, Cristiano RJ (1996) In vivo adenovirus-mediated p53 tumor
suppressor gene therapy for colorectal cancer. Anticancer Res 16,
3415-3422.
Steiner MS,
Lerner J, Greenberger M (1998)
Clinical phase I gene therapy trial using BRCA1 retrovirus is safe. J Urol 159, 133.
Steiner MS, Zhang
Y, Farooq F, Lerner J, Wang Y, Lu Y (2000)
Adenoviral vector containing wild type p16 suppresses prostate cancer growth
and prolongs survival by inducing cell senescence. Cancer Gene Ther 7, 360-372.
Stone S,
Dayananth P, Kamb A (1996)
Reversible p16-mediated cell cycle arrest as protection from chemotherapy. Cancer Res 56, 3199-3202.
Sutkowski DM,
Fong CJ, Sensibar JA, Rademaker AW, Sherwood ER, Kozlowski JM, Lee C (1992) Interaction of epidermal growth
factor and TGFb1 in human
prostatic epithelial cells in culture. Prostate
21, 133-143.
Talve L, Sauroja
I, Collan Y, Punnonen K, Ekfors T (1997) Loss of expression of the p16INK4/
CDKN2 gene in cutaneous malignant melanoma correlates with tumor cell
proliferation and invasive stage. Int J Cancer 74, 255-259.
Uhrbom L, Nister
M, Westermark B (1997) Induction of
senescence in human malignant glioma cells by p16INK4A. Oncogene 15, 505-514.
Walsh PC (1984) Benign prostatic hyperplasia:
Etiological considerations. In: Kimball FA, Buhl AE, Carter DE (eds).
Approaches to the Study of Benign Prostatic Hyperplasia, Liss: New York, pp
10-25.
Walsh PC (1984) Human benign prostatic
hyperplasia: etiological considerations. Prog
Clin Biol Res 145, 1-26.
Xio S, Li D, Vijg
J, Sugarbaker DJ, Corson JM, Fletcher JA (1995) Codeletions of p15 and p16 in
primary malignant mesothelioma. Oncogene 1, 511-515.
Xu M, Kumar D,
Srinivas S, Detolla LJ, Yu SF, Stass SA, Mixson AJ (1997) Parenteral gene therapy with p53 inhibits human breast tumors
in vivo through a bystander mechanism
without evidence of toxicity. Hum Gene
Ther 8, 177-185.
Yang C, Cirielli
C, Capogrossi MC, Passaniti A (1995)
Adenovirus-mediated wild-type p53 expression induces apoptosis and suppresses
tumorigenesis of prostatic tumor cells. Cancer
Res 55, 4210-4213.
Zhang XW, Qing C,
Xu B (1999) Apoptosis induction and
cell cycle perturbation in human hepatoma Hep G2 cells by
10-hydroxycamptothecin. Anticancer Drugs
10, 569-576.
