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Carcinogenesis |
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland 20892-4255
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The EGF-R is a membrane tyrosine kinase receptor that regulates multiple functions, including cell growth, differentiation, gene expression, and development (reviewed in Refs. 7 and 8 ). Keratinocytes express a number of EGF-like growth factors that function through autocrine, paracrine, and juxtacrine pathways. The EGF-R is frequently overexpressed in HPV-associated papillomas and carcinomas (9, 10, 11, 12) , and increased expression has been associated with poor prognosis in some (13, 14, 15, 16) but not all studies (11 , 17) . We have directly examined the importance of the EGF-R in HPV-associated tumorigenesis by using mice with a targeted disruption of the EGF-R gene (18) . Although this disruption causes strain-dependent embryonic lethality, strain CD-1 mice are born viable and survive for several days. Keratinocytes were cultured from the epidermis of neonatal CD-1 mice that were null, heterozygous, or wild-type for the EGF-R. Cultured cells were infected with retroviruses encoding the HPV-16 E6 and E7 genes, and the infected keratinocytes were grafted to the dorsal skin of nude mice to examine their tumorigenicity. Specifically, we examined whether the EGF-R was required for immortalization of keratinocytes by HPV-16 E6 and E7 genes, and whether cells lacking this receptor were able to progress to epidermal papillomas or squamous carcinomas.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Retrovirus Infection.
Primary mouse keratinocytes were inoculated into 12-well dishes
(2 x 104 cells/well) in EMEM
supplemented with KGF (1 ng/ml) to stimulate rapid proliferation.
Cultures were subsequently infected with recombinant retroviruses
encoding HPV-16 E6 and E7 genes (21)
by replacing the culture medium with supernatants from
retrovirus-producing cells for 3 h (22)
. Transduction
of HPV-immortalized cell lines with a replication defective retrovirus
encoding the v-ras oncogene was performed similarly
(19)
.
Immortalization Assay.
Keratinocytes were infected with HPV-16 retroviruses or with viruses
encoding only vector sequences as a control. Twenty-four h after
infection, cultures were trypsinized, replated from each well of a
12-well plate into a 1 x 60-mm dish, and maintained for
5 weeks. In selected experiments, cultures were maintained in medium
containing KGF (1 ng/ml) for the duration of the immortalization assay.
After 5 weeks, cultures were treated with 3% neutral-buffered formalin
for 30 min, rinsed three times in PBS, and stained with Giemsa to
visualize colonies. In some experiments, selected colonies were
subcultured to assess whether the cells were immortal. To establish
cell lines, all of the colonies from a single 1 x 60-mm
dish were pooled and subcultured.
Skin Grafts.
For grafting experiments, we used immortal keratinocyte cell lines that
had been passaged 6–12 times. Keratinocytes were grafted to
BALB/c-derived athymic nude mice between 2 and 3 months old. Immortal
keratinocytes (6 x 106)
were combined with 4 to 9 x 106
primary mouse fibroblasts, and the cell suspension (100 µl) was
inoculated beneath a silicon grafting chamber on the dorsal skin of
nude mice as described (20)
. In some experiments, grafts
were treated with 2 µg/25 µl TPA in acetone twice each week for 10
weeks. Animals were examined weekly, and tumors were measured using
calipers. Mice were euthanized when tumors were >2
cm3.
BrdUrd Labeling.
Mice were injected i.p. with 250 µg of BrdUrd/g of body weight in PBS
and sacrificed after 2 h. Unstained formalin-fixed paraffin
sections were placed in sodium citrate buffer and treated in the
microwave for 5 min to recover antigenicity. Slides were incubated with
PBS containing 10% normal goat serum and 3% BSA for 30 min before the
addition of anti-BrdUrd antibody (Becton Dickinson, Bedford, MA) for
2 h. After three washes in PBS, slides were treated with
biotinylated goat antimouse IgG (Jackson Laboratories, Bar Harbor, ME),
and specific staining was detected using a Vectastain ABC Elite Kit
(Vector Laboratories, Burlingame, CA).
Apoptosis.
The percentage of apoptotic keratinocytes was measured by adding
Hoecsht stain at a final concentration of 5 µg/ml for 10 min and
examining cultures by fluorescence microscopy. Nuclei that exhibited a
fragmented morphology and stained brightly were graded as apoptotic.
The percentage of apoptotic cells in skin grafts was determined using
the Apoptag Plus Kit (Intergen Company, Purchase, NY).
Western Blots.
Cultures that were 70–80% confluent were lysed at 4°C for 30 min in
PBS containing 1% NP40, 0.5% sodium lauryl sulfate, 0.1% SDS, and
protease inhibitors aprotinin, leupeptin, and phenylmethylsulfonyl
fluoride. Lysates were cleared by centrifugation and stored at -70°C
before use. Protein concentration was measured using the BCA Assay
(Pierce Chemical Co., Rockford, IL). Five to 10 µg of protein were
separated on 10% polyacrylamide gels and transferred to nitrocellulose
at 100 V for 1 h. The efficiency of transfer was confirmed by
staining membranes with Ponceau red. Membranes were incubated with 5%
nonfat milk overnight at 4°C to block nonspecific binding and then
incubated with primary antibodies (0.4 µg/ml for 1 h) to the
EGF-R or ErbB-2 (Santa Cruz Biotechnology, Santa
Cruz, CA). Membranes were incubated with antirabbit IgG horseradish
peroxidase conjugate (Bio-Rad, Hercules, CA) for 1 h, and
chemiluminescence was detected using X-ray film.
RT-PCR.
Subconfluent cultures of mouse keratinocytes were lysed with Trizol
(Life Technologies, Gaithersburg, MD), and the RNA was purified by
extraction with chloroform and precipitation in propanol. Residual DNA
was removed by precipitation in 2.5 M lithium chloride or
by DNase digest followed by another chloroform extraction and
isopropanol precipitation. For cDNA synthesis, 10 µg of total RNA
were reverse-transcribed for 1 h in the presence of 500
mM dNTPs, 1.25 mM oligo-dT primer, 40 units of
RNasin/ml, and 75 units/ml Superscript RTII (Life Technologies).
Reverse transcription reactions were diluted 50–100-fold for PCR, and
PCR amplifications (20 cycles) were performed in 20 µl using the
AmpliTaq Gold kit (Perkin-Elmer Applied Biosystems, Foster City, CA.).
PCR products were separated on 1.4% agarose gels and detected by
staining with ethidium bromide and UV transillumination.
cDNA Expression Arrays.
To create hybridization probes, purified RNA was reverse-transcribed in
the presence of either Cy3 or Cy5 dUTP-linked fluorescent dyes
(Amersham Pharmacia Biotech, Piscataway, NJ) plus 0.1 mg/ml of
oligo(dT) 20-mer primer, 0.5 mM each dATP, dGTP, and
dCTP, 0.2 mM dTTP, 0.01 mM DTT, 1x First
Strand Buffer (Life Technologies), 10 units of cloned RNasin (Life
Technologies), and 50 and 100 mg of RNA from EGF-R null and
EGF-R-positive cell lines, respectively. The RNA was
reverse-transcribed at 42°C for 60 min using 400 units of SuperScript
II with a spike of 400 additional units of enzyme after 25 min. The
reaction was stopped with 0.5 M EDTA, and the remaining RNA
was hydrolyzed with 167 mM NaOH. Reaction products were
diluted with Tris EDTA buffer (pH 7.4), concentrated to 20–40 ml in a
Microcon YM-30 centrifugal filter column, and combined to form the
hybridization mixtures. Each of the mixtures was a combination of probe
from one EGF-R null (Cy3) and one EGF-R wild-type or heterozygous (Cy5)
cell line. To each of the probe mixtures was added 10 mg of human COT-1
DNA, 10 mg of poly(A), 4 mg of yeast tRNA, 3.5x SSC, and 0.3% SDS.
These mixtures were denatured for 2 min at 100°C and cooled to room
temperature, 17.6 ml from each probe mixture was spotted on the surface
of a microarray, and a coverslip was applied. The arrays were allowed
to hybridize for 12 h at 65°C. Arrays were washed in 2x SSC
with 0.1% SDS to remove the coverslip, followed by 1-min washes in 1x
SSC, 0.2x SSC, and 0.05x SSC, and a quick rinse in distilled water.
The arrays were scanned on a Molecular Dynamics Avalanche scanner
(Amersham Pharmacia Biotech). The microarrays used were produced by the
core facility at the National Cancer Institute’s Advanced Technology
Center. Each contained target cDNA from 
2800 genes or expressed
sequence tags from the mouse genome.
Statistical Analyses.
Statistical comparisons using the t test and Fisher’s exact
test were performed with Sigma Stat statistical software (Jandel
Scientific, San Rafael, CA).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). E6/E7 expression induced 32 ± 6 colonies/60-mm dish
in +/+ or +/- cells, whereas no colonies were found in vector control.
In contrast, E6/E7 induced only 7 ± 2 colonies/60-mm
dish using EGF-R -/- cells. When cultures were continuously
supplemented with KGF, a mitogen associated with wound healing, colony
formation increased; however, the number of EGF-R -/- immortal
colonies was still less than EGF-R +/+ or +/- cultures. To confirm
that colonies consisted of immortal cells, 20 foci were picked and
subcultured. All grew continuously. These results suggest that the
EGF-R is important for efficient immortalization by HPV-16 E6 and E7,
but that this function may be partially bypassed by KGF.
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). The population doubling time of the EGF-R -/- cells was
significantly longer than +/- and +/+ cells (Table 1)
. The addition of exogenous KGF significantly decreased the doubling
time of both wild-type and null cells (data not shown). When cultures
were passaged extensively (15–20 passages), differences in doubling
time between EGF-R null and wild-type cells were not significant. We
routinely used cells between passage 6 and 12. EGF-R +/+ or +/- cells
continued to proliferate and pack together after they had reached
confluence, whereas null cells became quiescent or apoptotic (Fig. 2A
). Thus, the saturation density of EGF-R -/- immortal
keratinocytes maintained at confluence for 2 weeks was significantly
lower than the +/- and +/+ cells (Table 1)
. EGF-R -/- immortal
keratinocytes were also more susceptible to apoptosis when serum was
reduced in the culture medium (Table 1)
. Western blot analysis
confirmed that the EGF-R -/- cells failed to express the
Mr 170,000 EGF-R protein (Fig. 2B
). EGF-R +/+ and +/- lines expressed variable levels of
the receptor, and expression was not directly proportional to the gene
copy number. Levels of erbB-2, an EGF-R-related receptor,
were not altered in any of the genotypes. RT-PCR analysis showed that
the EGF-R -/-, +/-, and +/+ cell lines expressed similar levels of
E6/E7 and spliced E6*/E7 RNAs (Fig. 2C
). The same results
were observed using RNAs isolated from wild-type and null cells 1 week
after infection with E6/E7 retroviruses (before cells were clearly
immortalized). Thus, differences in the frequency of immortalization or
growth rate of immortal cells were not attributable to differences in
E6/E7 RNA expression.
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. EGF-R -/- cells expressed 5-fold less cyclin D RNA and 4- to 7-fold
higher levels of RNAs encoding growth inhibitory proteins
p21waf and IGFBP-2, which sequester IGF. EGF-R
-/- lines also expressed increased levels of several cytoskeletal
proteins, including profilin, desmoplakin, and tubulin, consistent with
their large flat morphology. Four transcription factors, erg-1, trop 2,
cysteine-rich protein 2, and E2A, were also increased in EGF-R -/-
cells.
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, EGFRH1–2 and EGFRWT1–2). Histological analysis confirmed that these
were benign papillomas (Fig. 3A
). Papillomas usually occurred within 6–10 weeks after
grafting. Some of the papillomas persisted, whereas others regressed.
Of the 90 grafts of EGF-R +/+ or +/- cells, 7 progressed to invasive
squamous cell carcinomas after 6–12 months (Fig. 3B
). In
contrast, grafts of EGF-R -/- immortal keratinocytes (EGFRKO-1 to -4)
did not form papillomas (Table 3)
. Histological analysis showed that
these grafts consisted of dysplastic epithelia with nests of
keratinocytes in the dermis (Fig. 3C
). Of the 96 grafts of
EGF-R null cells, 1 progressed to form a cystic carcinoma after 12
months. In summary, loss of the EGF-R prevented development of
papillomas and inhibited progression of grafted cells to carcinomas.
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. However, TUNEL staining revealed that apoptosis was not common in
grafts containing either EGF-R -/- or +/+ immortal keratinocytes
(data not shown).
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. Treatment with
TPA (2 µg/graft twice a week for 10 weeks) induced 4 tumors (2
carcinomas and 2 papillomas) in 22 grafts of EGF-R null immortal cells
after 8 months; no papillomas or carcinomas occurred in untreated
grafts (0 of 22). Treatment of grafts containing EGF-R-positive
keratinocytes induced 8 tumors (2 carcinomas and 6 papillomas) in 23
grafts, whereas 2 carcinomas occurred in untreated grafts after 8
months. These results suggest that TPA promotes malignant progression
of HPV-immortalized keratinocytes in part through an EGF-R-independent
pathway.
Mutant Ras Induces Carcinomas in E6/E7-immortalized
Keratinocytes Regardless of EGF-R Genotype.
Ras mediates signal transduction downstream of the EGF-R and
expression of a mutant ras gene stimulates malignant
progression of HPV-16-immortalized keratinocytes (4)
. We
examined whether loss of the EGF-R would make immortalized
keratinocytes less susceptible to malignant transformation after
infection with retroviruses encoding v-Ha-ras. Four
different EGF-R -/- cell lines expressing ras grew rapidly
in culture and reached a high saturation density similar to EGF-R
wild-type cells. Immortal keratinocytes expressing activated
ras formed rapidly growing carcinomas in all grafts
regardless of EGF-R genotype (Table 3
and Fig. 3D
). Although
tumor growth rates varied among the eight ras-infected cell
lines (data not shown), there were no significant differences between
EGF-R -/- and +/+ or +/- genotypes. Thus, ras activation
bypasses the requirement for the EGF-R in malignant transformation of
HPV-immortalized keratinocytes.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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EGF-R null keratinocytes were immortalized less efficiently by HPV-16 E6 and E7 genes than wild-type or heterozygous cells. RT-PCR analysis indicated that immortal cells of each genotype expressed similar levels of E6/E7 and spliced E6*/E7 RNAs, indicating that differences in virus oncoprotein expression could not account for differences in immortalization efficiency. Cultures of EGF-R null immortal keratinocytes grew more slowly, exhibited decreased saturation density, and were more susceptible to apoptosis. Efficient immortalization by E6 and E7 depends on genetic alterations that occur during crisis and propagation of cells in culture (26) . The decreased rate of proliferation and enhanced susceptibility to apoptosis of EGF-R -/- keratinocytes might render these cells less able to survive crisis and progress to full immortalization. The addition of KGF, which increases mitogenesis and inhibits apoptosis (27) , bypassed the requirement for the EGF-R. Thus, the EGF-R is important but not necessary for immortalization by HPV-16 E6 and E7.
HPV-immortalized keratinocytes that lacked the EGF-R did not develop
epidermal papillomas when grafted with normal fibroblasts. In contrast,
wild-type and heterozygous cells formed papillomas in 17% of grafts.
The latter observation suggests that immortalization by E6 and E7 is
not alone sufficient to induce papillomas or carcinomas. These findings
are consistent with reports that expression of HPV-16 E6 and
E7 genes in the epidermis of transgenic mice induces
papillomas and carcinomas after a long latent period (28
, 29)
. Most interestingly, our results provide the first direct
evidence that the EGF-R is important for the progression of
HPV-immortalized keratinocytes to papillomas and squamous
carcinomas. Previous studies have shown that the EGF-R serves an
important function in mouse epidermal carcinogenesis. The EGF-R is
overexpressed in chemically induced skin tumors (30)
, and
transgenic mice that produce increased levels of transforming growth
factor 
have a greater incidence of epidermal carcinomas after
treatment with chemical carcinogen (31)
. Expression of the
EGF-R and EGF-R signaling are increased in HPV-infected human laryngeal
papilloma cells (10)
. Signaling through the EGF-R is
required for mitogenic stimulation of HPV-immortalized keratinocytes by
proinflammatory cytokines (32
, 33)
. Furthermore,
HPV-immortalized keratinocytes exhibit constitutive activation of the
EGF-R in vitro (34)
, and pharmacological
inhibition of EGF-R function effectively blocks growth and survival of
immortal keratinocytes in culture (35
, 36)
. Our results
extend these observations to an in vivo model and suggest
that inhibitors of EGF-R function, such as blocking antibodies and
kinase inhibitors, may be useful for stimulating apoptosis or blocking
cell growth of HPV-associated papillomas. Previous work has shown that
monoclonal antibodies that block binding to the EGF-R can sensitize
carcinoma cells to chemotherapeutic agents (37)
, and can
eradicate established tumors growing as xenografts in mice
(38)
.
Our results indicate that the EGF-R is important but not necessary for immortalization of mouse keratinocytes by HPV or for progression to malignancy. These results should be interpreted with the understanding that human keratinocytes may behave differently than mouse cells. Furthermore, we overexpressed the E6 and E7 genes from a retrovirus long terminal repeat rather than from the endogenous HPV upstream regulatory region. Overexpression of E6 and E7 might partially bypass a requirement for the EGF-R in immortalization or tumorigenesis at lower levels of virus gene expression.
The EGF-R activates multiple signaling pathways that may contribute to malignant development and progression (7 , 8) . Our in vivo and in vitro analyses indicate that EGF-R null immortal keratinocytes have reduced proliferative potential. Although the EGF-R -/- cells were more susceptible to apoptosis in vitro, increased apoptosis was not detected in EGF-R -/- grafts using TUNEL staining. However, EGF-R -/- cells in grafts could be lost by necrosis or through an apoptotic pathway that does not cause the usual pattern of DNA fragmentation. Others have shown that pharmacological inhibition of EGF-R function stimulates apoptosis and squamous differentiation in HPV-immortalized human keratinocytes in vitro (35) .
We used cDNA arrays to examine differences in gene expression between
EGF-R null and wild-type HPV-immortalized keratinocytes that might
contribute to decreased tumorigenicity. We observed multiple
alterations in gene expression (3–5% of genes on arrays) in any one
comparison of wild-type and null immortal cells. However, many of these
changes did not appear to specifically relate to loss of the EGF-R. It
is possible that immortalization is accompanied by diverse changes in
gene expression; thus, any comparison between two immortal lines will
identify both EGF-R-specific and -nonspecific changes. In fact, <1%
of genes on arrays were reproducibly up- or down-regulated 
3-fold in
three of four independent comparisons between EGF-R -/- and +/+ or
+/- lines. EGF-R null keratinocytes expressed reduced levels of cyclin
D RNA and increased levels of RNAs encoding the cyclin-dependent kinase
inhibitor p21waf. Consistent with our findings,
inhibition of EGF-R tyrosine phosphorylation in HPV-immortalized
keratinocytes results in large increases in
p21waf and p27kip
(35)
. The largest alteration in gene expression that we
observed in EGF-R -/- cells was a 7-fold increase in RNA for the
IGFBP-2 that binds and sequesters IGFs. Decreased availability of IGFs
is potentially significant because activation of the IGF-1 receptor
enhances susceptibility to diverse skin tumor promoters and may play an
important role in epidermal carcinogenesis (39)
. The IGF-1
receptor is required for immortalization of fibroblasts by HPV-16 E7
(40)
, and IGF-2 mediates EGF-induced mitogenesis in
cervical cancer cells (41)
. Our findings identify specific
alterations in expression of cellular genes resulting from a targeted
disruption of the EGF-R gene. However, it is unclear whether
these changes or possibly others that were not identified on arrays are
critical for inhibition of papilloma growth and malignant progression.
Previous studies have shown that the tumor promoter TPA induces expression of EGF-R ligands and increases tyrosine phosphorylation of the EGF-R, which implies an important role for EGF-R signaling in tumor promotion of mouse skin (42) . Our results show that topical application of TPA induced papillomas and carcinomas in grafts of EGF-R null immortal keratinocytes. These results provide direct evidence that TPA may also promote epidermal carcinogenesis through an EGF-R-independent pathway.
Ras mediates signaling downstream of the EGF-R and activates
multiple pathways that influence cell growth and transformation
(43)
. Our results show that expression of an activated
ras gene can bypass the requirement for the EGF-R and induce
rapidly growing carcinomas regardless of EGF-R genotype. Activation of
ras is important for HPV-associated carcinogenesis.
Mutations in ras occur in a subset of cervical cancers
(44)
, and an activated ras gene induces
malignant conversion of HPV-immortalized human keratinocytes
(4)
. Our results suggest that the EGF-R may not be
critical for transformation of HPV-infected cells that have
ras mutations. Interestingly, normal keratinocytes from
EGF-R null mice form papillomas less efficiently than wild-type cells
after the introduction of activated ras (19)
.
Furthermore, activation of the EGF-R by TGF- promotes skin
tumorigenesis by ras-independent pathways (31)
.
This suggests that the EGF-R stimulates pathways that are distinct from
and complement ras functions. In contrast, these alternate
pathways may already be activated or they may be less important in
keratinocytes immortalized by HPV-16 E6 and E7.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Biology Department, Clarkson University, Potsdam, NY
13699. Phone: (315) 268-2391; Fax: (315) 268-7118; E-mail: woodworth{at}clarkson.edu 
2 The abbreviations used are: HPV, human
papillomavirus; EGF, epidermal growth factor; EGF-R, EGF receptor; IGF,
insulin-like growth factor; KGF, keratinocyte growth factor;
BrdUrd, 5-bromo-2'-deoxyuridine; EMEM, Eagles’ minimal
essential medium; TPA,
12-O-tetradecanoylphorbol-13-acetatel; RT-PCR, reverse
transcription-PCR; TUNEL, terminal deoxynucleotidyl
transferase-mediated nick end labeling; IGFBP2, IGF-binding protein
2.
Received 12/ 8/99. Accepted 6/19/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and tumor necrosis factor stimulate autocrine amphiregulin expression and proliferation of human papillomavirus-immortalized and carcinoma-derived cervical epithelial cells. Proc. Natl. Acad. Sci. USA, 92: 2840-2844, 1995.This article has been cited by other articles:
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  D. Brennan, Y. Hu, S. Joubeh, Y. W. Choi, D. Whitaker-Menezes, T. O'Brien, J. Uitto, U. Rodeck, and M. G. Mahoney Suprabasal Dsg2 expression in transgenic mouse skin confers a hyperproliferative and apoptosis-resistant phenotype to keratinocytes J. Cell Sci., March 1, 2007; 120(5): 758 - 771. [Abstract] [Full Text] [PDF]
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 S. J.S. Simonson, M. J. Difilippantonio, and P. F. Lambert Two Distinct Activities Contribute to Human Papillomavirus 16 E6's Oncogenic Potential Cancer Res., September 15, 2005; 65(18): 8266 - 8273. [Abstract] [Full Text] [PDF]
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S. M. Genther Williams, G. L. Disbrow, R. Schlegel, D. Lee, D. W. Threadgill, and P. F. Lambert Requirement of Epidermal Growth Factor Receptor for Hyperplasia Induced by E5, a High-Risk Human Papillomavirus Oncogene Cancer Res., August 1, 2005; 65(15): 6534 - 6542. [Abstract] [Full Text] [PDF]
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 C. D. Woodworth, E. Michael, D. Marker, S. Allen, L. Smith, and M. Nees Inhibition of the epidermal growth factor receptor increases expression of genes that stimulate inflammation, apoptosis, and cell attachment Mol. Cancer Ther., April 1, 2005; 4(4): 650 - 658. [Abstract] [Full Text] [PDF]
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 M C Velarde, S I Parisek, R R Eason, F A Simmen, and R C M Simmen The secretory leukocyte protease inhibitor gene is a target of epidermal growth factor receptor action in endometrial epithelial cells J. Endocrinol., January 1, 2005; 184(1): 141 - 151. [Abstract] [Full Text] [PDF]
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