Department of Pathology and Immunology, and Division of Molecular Oncology in the Department of Internal Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA
e-mail: kathryn.wikenheiser-brokamp{at}uc.edu
Accepted 23 April 2004
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SUMMARY |
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Key words: Rb, p107, p130, Cre-LoxP system, Lung development, CC10
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Introduction |
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Cellular sensitivity to Rb deficiency may reflect differing degrees of
functional redundancy among family members in distinct cell types. Rb is a
member of a protein family that also includes p107 and p130. These three
family members share extensive structural homology, especially in a bipartite
pocket domain that functions as the binding site for viral oncoproteins and
acts as a transcriptional repressor motif
(Classon and Dyson, 2001). All
three pocket proteins inhibit E2F responsive promoters, recruit chromatin
remodeling enzymes, actively repress transcription, and growth arrest cells
when overexpressed (Classon and Dyson,
2001
; Harbour and Dean,
2000
). These structural and biochemical similarities probably
explain the functional overlap among pocket proteins during development, and
provide rationale for the observation that p107 and p130 can at least
partially compensate for loss of Rb function in vivo
(Lipinski and Jacks, 1999
). An
important distinction among the pocket proteins, however, is that Rb, but not
p107 and p130, has been shown to be a tumor suppressor in humans
(Classon and Dyson, 2001
).
Defining the overlapping versus distinct functions of Rb family proteins is
therefore an important step to understanding the unique role of Rb.
The lung provides a manipulatable model system in which to explore Rb
family function in vivo, both in cell cycle control and in cellular
differentiation. The respiratory epithelium comprises markedly diverse and
specialized cell types that are derived from a common progenitor population.
Cellular proliferation and differentiation are coordinately regulated to
create this diversity, which is required for normal lung function
(Kauffman, 1980). Rb, p130 and
p107 are all expressed in the developing and adult lung. p107 expression is
fairly uniform in the developing lung, with levels peaking during midgestation
(embryonic day 12.5-13.5) and then declining thereafter
(Jiang et al., 1997
;
Kim et al., 1995
). By
contrast, p130 and Rb expression increase during late embryonic development
and are maintained at relatively high levels in the adult lung
(Bernards et al., 1989
;
Chen et al., 1996a
;
Garriga et al., 1998
;
Jiang et al., 1997
;
Levine et al., 1998
;
Pertile et al., 1995
).
Furthermore, accumulation of the active, hypophosphorylated form of Rb is
coincident with epithelial cell differentiation and growth arrest
(Levine et al., 1998
). Taken
together, these data suggest that pocket protein function is important for
lung development.
Rb has also been implicated as a crucial tumor suppressor in the lung
epithelium. Lung carcinomas are divided into small cell (SCLC) and non-small
cell lung cancers (NSCLC), based upon distinct clinical and pathologic
features. Rb gene mutations are found in nearly all SCLC, an aggressive
neoplasm that shares a neural phenotype with retinoblastoma
(Kaye, 2001;
Minna et al., 2002
). The
nearly universal and selective occurrence of Rb gene mutations in SCLC
provides evidence that Rb loss is essential in the genesis of this malignancy,
and suggests that specific cell types within the lung epithelium are
particularly sensitive to loss of Rb function.
The current studies were designed to determine putative cell-type-specific functions of Rb that are not functionally redundant with other family proteins. The results provide convincing evidence that pocket protein function is essential for lung epithelial development. Unexpectedly, pocket proteins also have opposing roles in specification along distinct cell lineages; they inhibit neuroendocrine cell fate but are required for differentiation in other cell types. Remarkably, Rb is specifically required for restricting neuroendocrine cell fate despite functional compensation for Rb deficiency in other cell types. The selective occurrence of Rb gene mutations in human SCLC correlates well with the cell lineage-specific functions for Rb seen in the currently generated mouse model. Thus, these studies demonstrate that Rb acts as a cell-specific regulator in epithelial development and provide a mouse model that mimics human disease associated with Rb deficiency. The results also identify and define a novel role for pocket proteins in cell lineage specification.
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Materials and methods |
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Doxycycline administration
Gestation was dated by detection of vaginal plug (E0.5). Dams were treated
with 125 µg doxycycline (Sigma) in 0.5 ml PBS by intraperitoneal injection
on E0.5-E1.5 and administered doxycycline in the drinking water at a final
concentration of 1.0 mg/ml. A 50x doxycycline stock solution (50 mg/ml
in 50% ethanol) was freshly prepared prior to each administration of drug and
diluted in water or in PBS for injection. Doxycycline water was replaced three
times per week because of the light sensitivity of doxycycline.
Histology, immunohistochemistry and TUNEL analysis
Lung tissue was fixed in 10% neutral buffered formalin and paraffin
embedded. Sections were prepared and stained with Hematoxylin and Eosin for
histological analysis. Immunohistochemistry was performed using Vectastain
Elite ABC, M.O.M. Immunodetection, and DAB Substrate Kits (Vector
Laboratories). Methanol/hydrogen peroxide pre-treatment and microwave/10 mM
citrate antigen retrieval were performed. Antibodies were diluted in 0.1%
bovine serum albumin in PBS, applied to sections and incubated overnight at
4°C. Antibodies and dilutions used were as follows: SV40 T Ag Ab-2, 1:80
(Oncogene); PCNA PC10, 1:200 (PharMingen); Ki67, 1:50 (BD PharMingen); CGRP,
1:10,000; (Sigma); and CCSP and Foxj1, 1:20,000 and 1:500, respectively
(kindly provided by Steve Brody, Washington University, St Louis, MO, USA).
TUNEL analysis was performed on sections using ApoTag Peroxidase Detection Kit
(Intergen). Slides were counterstained with Hematoxylin. Quantitation of
apoptosis and proliferation was carried out by counting the number of TUNEL-
or Ki67-positive cells, respectively. Percentages were determined by
evaluating 400 cells representing proximal and distal conducting airways and
at least two lung lobes per mouse. Quantitation of neuroendocrine cell foci
was performed by counting CGRP-positive foci per mm of airway. Counts
represent analysis of 8.5-10 mm of airway and at least two lung lobes per
mouse. Littermates were used as controls for all studies when possible.
Statistical significance was determined by Student's t-test.
Laser capture microdissection (LCM)
Five micron sections of formalin-fixed, paraffin-embedded tissue were
placed on uncharged, non-coated glass slides, dried and put into a dessicator.
Slides were deparaffinized, rinsed in distilled water and stained in
Hematoxylin for 30 seconds and Eosin for 11 seconds. LCM was performed with
the Pixcell II Laser Capture Microdissection and Image Archiving Workstation
systems (Arcturus Engineering). Epithelial cells were captured on
thermoplastic caps and digested in proteinase K digestion buffer at 55°C
overnight. Samples were heated at 95°C for 15 minutes to inactivate the
protease and used directly for PCR analysis. The Rb alleles were analyzed
using two separate primer sets: (1) primers Rb-18 and Rb-19 that yield
products of 678 bp for the wild-type and Rb knockout alleles, 746 bp for the
floxed Rb allele and 321 bp for the recombined Rb allele
(Vooijs et al., 2002); and (2)
primers Rb-212, Rb-18 and Rb-19E that yield products of 235 bp for the
wild-type and Rb knockout alleles, 283 bp for the floxed Rb allele and 260 bp
for the recombined Rb allele (Meuwissen, 2003).
ß-Galactosidase staining
Whole-mount and frozen section staining for ß-galactosidase activity
was performed as described (Nagy, 2003). Briefly, whole lung was fixed in 0.4%
paraformaldehyde, rinsed in detergent buffer and incubated at 37°C
overnight in stain solution. For section staining, tissue was fixed in 0.2%
paraformaldehyde, cryoprotected in 30% sucrose and embedded in OCT compound.
Three or five micron sections were postfixed in 0.2% paraformaldehyde, rinsed
in saline and detergent buffers and incubated at 37°C overnight in stain
solution. Slides were subsequently counterstained with Nuclear Fast Red or
used in a modified immunoassay for CGRP, wherein methanol/hydrogen peroxide
pre-treatment and antigen retrieval were eliminated, and antibody was
incubated at room temperature for 1 hour.
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Results |
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Rb regulates epithelial cell proliferation and survival during development
Lungs from double-transgenic mice treated with doxycycline (hereafter
referred to as CC10-rtTA) were grossly normal and showed normal branching
morphogenesis. Microscopic analysis, however, showed marked epithelial cell
abnormalities throughout the conducting airways. The epithelium was
hypercellular and composed of dysplastic cells with increased nuclear to
cytoplasmic ratios and cells containing pyknotic nuclei
(Fig. 2A). These epithelial
changes were not present in the absence of doxycycline treatment
(Fig. 2B), or in control
littermates lacking one or both transgenes required for Rb gene ablation
(Fig. 2C). The phenotype was
similar in double transgenic mice with RbLoxP/LoxP or
RbLoxP/- alleles, and therefore results shown for all analyses are
representative of both genotypes.
|
Rb ablation leads to increased proliferating cell nuclear antigen (PCNA) expression
To confirm that Rb function is indeed lost in the lung epithelium of
double-transgenic mice treated with doxycycline, immunohistochemical analysis
was performed for the E2F-regulated gene product PCNA. Rb/E2F complexes
repress gene transcription (Harbour and
Dean, 2000). Therefore, loss of Rb function would be expected to
lead to derepression of E2F-regulated genes and thus increased PCNA
expression. Immunohistochemical analysis demonstrated uniform PCNA expression
throughout the conducting airways in CC10-rtTA lungs
(Fig. 2J). Derepression of PCNA
was dependent upon Rb ablation, as increased PCNA expression was not detected
in controls lacking one or both transgenes, or in the absence of doxycycline
(Fig. 2K,L). These data provide
evidence that Rb function is lost in the vast majority of epithelial cells
upon doxycycline treatment.
Rb loss selectively effects neuroendocrine cell fate during development
Rb gene mutations are preferentially found in neuroendocrine versus
non-neuroendocrine lung carcinomas (Kaye,
2001; Minna et al.,
2002
), implying that Rb has cell lineage-specific functions. To
explore this possibility, epithelial differentiation was assessed in CC10-rtTA
lungs by morphology, along with immunohistochemical analysis for markers of
Clara cell [Clara cell specific protein (CCSP)] and neuroendocrine cell (CGRP)
differentiation. Clara and ciliated cell differentiation was similar in
Rb-deficient and control lungs (Fig.
3A-C, and data not shown). Interestingly, however, Rb-deficient
lungs showed an increase in neuroendocrine cells within the airway
(Fig. 3D-F).
Immunohistochemical analysis for CGRP showed scattered single cells and small
cell aggregates predominantly at airway branchpoints in control lungs,
consistent with the location of pulmonary neuroendocrine cells. By contrast,
Rb-deficient lungs showed an increase in CGRP immunoreactive epithelial
aggregates. Quantification of CGRP immunoreactive foci per mm of airway showed
a statistically significant increase in CC10-rtTA mice, when compared with
controls lacking one or both transgenes required for Rb ablation, and lungs
from mice not treated with doxycycline [2.3±0.7 versus 1.4±0.5
(P<0.02) for control and 1.6±0.3 (P<0.05) for
No Dox lungs]. By contrast, there was no statistically significant difference
between untreated mice and controls lacking one or both transgenes. Thus, Rb
is not essential for non-neuroendocrine cell differentiation. Instead, Rb
specifically restricts the neuroendocrine cell lineage.
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|
Rb deficiency leads to hypercellular neuroendocrine lesions
Although the epithelium in adult CC10-rtTA mice was remarkably restored in
comparison with lungs from day 1 pups, neuroendocrine cell abnormalities were
present. Lungs from adult mice showed multifocal hypercellular epithelial
lesions located predominantly at airway branchpoints and bronchiolo-alveolar
duct junctions (Fig. 4A-C).
These lesions were composed of epithelial cells showing high nuclear to
cytoplasmic ratios that protruded into the airway lumens. Sloughed cellular
aggregates were also noted within airway lumens. The cells exhibited a
neuroendocrine phenotype, as evidenced morphologically and confirmed by CGRP
expression (Fig. 4C). Thus, Rb
has a cell lineage-specific role that is essential for regulation of
neuroendocrine cell fate.
Rb family function is essential for lung epithelial development and neonatal survival
Compensation for loss of Rb function in non-neuroendocrine cells could be
due to functional redundancy with other Rb family proteins, namely p107 and
p130. To test this possibility, transgenic mice were created wherein total Rb
family function was ablated in a lung epithelial specific manner. A truncated
SV40 large T antigen oncoprotein (T121) that binds and specifically perturbs
pocket protein function (Symonds et al.,
1994) was targeted to the developing lung epithelium using
promoters from the rat CC10 (Stripp et
al., 1992
) and the human surfactant protein C (SPC)
(Glasser et al., 1991
) genes.
Although endogenous SPC is expressed in distal Type II cells and CC10 is
confined to Clara cells in the adult mouse lung
(Zhou et al., 1996
), both
promoters direct transgene expression to progenitor cells in the lung
epithelium early in development (embryonic day 10-12.5), before
differentiation into specialized cell types
(Hackett and Gitlin, 1994
;
Wert et al., 1993
). The CC10
promoter directs expression throughout the developing epithelium, whereas
transgene expression directed by the SPC promoter is confined to progenitor
cells that give rise to the distal respiratory airway. To ensure the phenotype
is dependent upon loss of pocket protein function, transgenic mice were also
generated with the same promoters driving expression of the truncated SV40
large T antigen containing a single base pair mutation (TK1) that is known to
disrupt Rb family binding (Symonds et al.,
1994
).
Potential transgenic founder animals screened at the time of weaning showed a significantly lower percentage of positive animals with T121 versus the control TK1 transgene (Table 2). Additionally, surviving CC10-T121 and SPC-T121 founders did not express the transgene (data not shown). These results suggest that Rb family ablation resulted in lethality prior to weaning. To address this possibility, potential CC10-T121 founder animals were delivered by Caesarean section one day prior to expected delivery as lung function is first required at birth. Seventy-five percent (9/12) of transgene-positive pups died within the first 15 minutes after Caesarean delivery because of respiratory failure characterized by irregular gasping and failure to establish a regular breathing pattern and pink color. Surviving transgenic founders maintained using foster mothers did not express the transgene. These results indicate that Rb family function in the lung epithelium is essential for neonatal survival.
|
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|
Loss of Rb family function leads to increased expression of PCNA
To confirm that pocket protein function is lost in the epithelium of
CC10-T121 lungs, immunohistochemical analysis was performed for the
E2F-regulated gene product PCNA. Indeed, PCNA was detected throughout the
epithelium in CC10-T121 lungs, which is in marked contrast to the scattered
immunopositive cells observed in wild-type lungs
(Fig. 5I,J). These data provide
evidence that Rb family function is lost in CC10-T121 lungs.
Ablation of Rb family function blocks cellular differentiation along both Clara and ciliated cell lineages
Morphological characteristics typical of Clara and ciliated cell lineages
were lacking in CC10-T121 transgenic lungs indicating impaired cellular
differentiation. To further investigate cell lineage specification,
immunohistochemistry was performed for markers of Clara cell (CCSP)
(Zhou et al., 1996) and
ciliated cell [hepatocyte nuclear factor-3/forkhead homolog 4 (HFH-4/Foxj1)]
(Blatt et al., 1999
;
Tichelaar et al., 1999
)
differentiation. CCSP and Foxj1 are expressed in the respective cell types
prior to the appearance of morphologic indicators of differentiation. CCSP and
Foxj1 expression were lacking or markedly reduced in CC10-T121 transgenic
lungs (Fig. 7A and data not
shown).
|
Ablation of Rb family function leads to an increase in neuroendocrine cells
Neuroendocrine cell differentiation was assessed by immunohistochemical
analysis for CGRP expression. CC10-T121 transgenic lungs showed an increase in
the number of neuroendocrine cell aggregates when compared with wild-type
controls (Fig. 7D,E,H).
Additionally, larger neuroendocrine cell aggregates were found in transgenic
lungs when compared with wild-type control lungs (18% of aggregates were
composed of >10 cells in CC10-T121 lungs versus 4% in wild-type lungs;
P=0.026). Thus, the data support cell lineage-specific functions for
Rb family proteins in the airway epithelium; acting as essential promoters of
nonneuroendocrine cell differentiation while suppressing neuroendocrine cell
fate. Interestingly, lungs with only Rb deficiency show
neuroendocrine-specific abnormalities. Taken together, these results provide
strong evidence that Rb has a cell lineage-specific role that is essential for
the regulation of neuroendocrine cell fate despite family member compensation
for Rb deficiency in non-neuroendocrine cell lineages.
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Discussion |
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Rb provides a unique cell lineage-specific function in epithelial development
The phenotypes of Rb and total pocket protein-deficient lungs show that
pocket proteins are essential in lung epithelial development for the
regulation of epithelial cell proliferation, survival and differentiation.
Specifically, pocket proteins augment cell survival and non-neuroendocrine
differentiation, while suppressing proliferation and neuroendocrine cell fate.
It remains possible that the T121 transgene used in the present studies may
alter cellular functions in addition to Rb family function. Nevertheless, the
lack of phenotypic abnormalities in control mice expressing the same transgene
with a single base pair mutation known to eliminate Rb family binding provides
evidence that pocket protein function is essential during epithelial
development.
The current studies provide direct evidence that Rb has a unique and
essential role in negatively regulating neuroendocrine cell fate in vivo. The
epithelial-specific Rb knockout model generated in the current work
demonstrates that Rb itself is capable of suppressing proliferation and
enhancing cell survival during epithelial development. Interestingly,
compensation for loss of Rb function occurred with regard to these functions,
enabling restoration of the airway epithelium. By contrast, Rb function was
absolutely required for regulating the neuroendocrine cell lineage. This
unique cell lineage-specific function of Rb is not only essential in
development but also in tumor suppression, as Rb ablation led to hypercellular
neuroendocrine lesions in the current study, and somatic inactivation of both
Rb and p53 in the mouse lung induces small cell lung cancers
(Meuwissen et al., 2003).
Furthermore, the observation that Rb is specifically, and nearly universally,
mutated in human SCLC suggests that this unique cell lineage-specific Rb
function is also important in the suppression of human malignancies.
Rb family proteins have opposing roles in differentiation along distinct cell lineages
The present work provides evidence that pocket protein function is crucial
for restricting neuroendocrine cell fate while promoting differentiation in
other cell types. There are three mechanisms by which pocket protein
inactivation could lead to an increase in neuroendocrine cells: (1) decreased
apoptosis, (2) increased proliferation, or (3) increased differentiation.
Decreased apoptosis is unlikely as there is no evidence that neuroendocrine
cell number is regulated by apoptosis in normal lung development, and
apoptosis was not detected in wild-type lungs in the present studies.
Furthermore, loss of pocket protein function has been shown to induce rather
than inhibit apoptosis (Dannenberg et al.,
2000; Lipinski and Jacks,
1999
; Sage et al.,
2000
). Enhanced neuroendocrine cell proliferation is the most
obvious mechanism. Although increased proliferation would explain an overall
increase in neuroendocrine cell number, this mechanism does not account for
the increase in neuroendocrine foci seen in pocket protein-deficient lungs.
Moreover, the majority of neuroendocrine cells in CC10-T121 transgenic lungs
are not immunoreactive for the proliferation marker Ki67 in co-localization
studies (data not shown). The prominence of neuroendocrine cells in pocket
protein-deficient lungs is therefore not likely to occur simply as a result of
increased cellular proliferation. Thus, the data suggest that loss of Rb
family function leads to increased neuroendocrine differentiation.
Although pocket proteins have previously been shown to augment
differentiation (Chen et al.,
1996b; Gu et al.,
1993
; Novitch et al.,
1996
; Thomas et al.,
2001
), Rb has not previously been implicated in suppressing a
differentiation pathway. Interestingly, SCLC and retinoblastomas share a
neural phenotype and are the only human malignancies that exhibit Rb gene
mutations in nearly all cases (Sherr,
1996
). Rb+/- mice and chimeric animals made from
Rb-/- ES cells also develop neuroendocrine malignancies
(Hu et al., 1994
;
Jacks et al., 1992
;
Maandag et al., 1994
), albeit
not the same tumors associated with Rb loss in humans. In addition, ES cells
lacking pocket protein function show exclusive neural differentiation, whereas
normal totipotent ES cells differentiate along endodermal, ectodermal and
mesodermal cell lineages (Dannenberg et
al., 2000
). Taken together, these data suggest a novel role for Rb
in suppression of differentiation. Moreover, the mechanisms underlying Rb
function in cellular differentiation and tumor suppression are likely to be
linked, given the strong association between Rb loss and neuroendocrine
differentiation in tumors.
Functional redundancy among pocket proteins provides an explanation for why Rb and p16 are differentially targeted in phenotypically distinct carcinomas
Lung carcinomas are divided into SCLC and NSCLC, based upon distinct
clinical and pathologic features. Rb gene mutations occur in nearly all SCLC,
whereas p16 is the preferential target for inactivation in NSCLC
(Kaye, 2001;
Minna et al., 2002
). The p16
protein inhibits cyclin D/cdk4,6 kinase activity thus maintaining Rb in its
active, hypophosphorylated state. Inactivation of p16 occurs in many human
cancers and results in constitutive hyperphosphorylation and thus inactivation
of Rb (Sherr and McCormick,
2002
). The remarkably tight inverse correlation between mutational
inactivation of Rb and loss of p16 expression suggest that these proteins
function in a common regulatory pathway
(Otterson et al., 1994
;
Shapiro et al., 1995
). Why
then are different components of the Rb pathway selectively mutated in
distinct carcinomas? One hypothesis is that Rb gene mutations are seen in SCLC
because neuroendocrine cells are exquisitely sensitive to Rb loss because of a
lack of functional compensation by p107 and/or p130 in this cell lineage. By
contrast, Rb mutations are not detected in NSCLC because these tumors arise
from nonneuroendocrine cell lineages
(Minna et al., 2002
) that show
functional compensation for Rb deficiency. In support of this hypothesis, Rb
function was demonstrated to be specifically required for regulation of
neuroendocrine but not other epithelial cell lineages in the current studies.
Total pocket protein inactivation resulted in marked epithelial abnormalities
throughout the epithelium, implying that p107 and/or p130 provide a redundant
or compensatory function in other cell lineages. Inactivation of p16 alters
total pocket protein function, thereby eliminating family member compensation
that occurs with Rb loss alone (Classon and
Dyson, 2001
; Sherr and
McCormick, 2002
; Tedesco et
al., 2002
). Moreover, p107 or p130 is required along with Rb for
p16-mediated growth arrest in mouse embryo fibroblasts
(Bruce et al., 2000
). Thus, the
data support the hypothesis that Rb gene mutation is sufficient to generate
SCLC but that p16 inactivation is required to generate NSCLC because of
differing degrees of functional redundancy among pocket proteins in distinct
cell types. In human cancers, p16 inactivation occurs with much greater
frequency than Rb gene mutations (Sherr,
1996
) suggesting that, in contrast to lung neuroendocrine cells
and retinoblasts, most cells require loss of total pocket protein function
rather than simply Rb to progress to cancer.
Rb deficiency in the mouse lung epithelium mimics human disease
Rb+/- mice develop pituitary and thyroid tumors but unexpectedly
do not develop retinoblastoma (Hu et al.,
1994; Jacks et al.,
1992
; Maandag et al.,
1994
; Williams et al.,
1994
). Furthermore, genetically altered mice with Rb-deficient
photoreceptor cells show no retinal abnormalities, even in a p53 null
background (Vooijs et al.,
2002
). Importantly, the mouse retina is not intrinsically
resistant to the development of retinoblastoma as this tumor occurs in
transgenic mice expressing viral oncoproteins in photoreceptor cells
(Vooijs and Berns, 1999
). The
striking discordance between the development of retinoblastomas in humans with
Rb germline mutations versus mice has raised the general question as to
whether engineered mice can be used to model human disease resulting from Rb
deficiency.
The lung phenotype seen upon conditional Rb gene activation in the current
studies shows similarities with lung disease resulting from loss of Rb
function in humans. First, the nearly universal and selective occurrence of Rb
gene mutations in neuroendocrine as opposed to non-neuroendocrine human lung
carcinomas correlates well with the hypercellular neuroendocrine lesions
observed after Rb ablation in the mouse. Second, germline Rb mutations in
humans would not be predicted to lead to global lung abnormalities, based on
the remarkable compensation for loss of Rb function seen in the mouse lung.
However, germline Rb mutations would be predicted to predispose individuals to
neuroendocrine tumors, specifically to SCLC. Indeed, multiple studies have now
established that germ line Rb mutations in humans confer an increased risk to
lung cancer (Kleinerman et al.,
2000; Leonard et al.,
1988
; Sanders et al.,
1989
; Strong et al.,
1984
). Epidemiological studies show that carriers of a mutant Rb
allele are 15 times more likely to die from lung cancer than the general
population (Sanders et al.,
1989
). Moreover, the tumors that arise in these patients are
predominantly SCLC and develop at a younger age than in the general population
(Leonard et al., 1988
;
Sanders et al., 1989
;
Strong et al., 1984
). The
mouse model generated for these studies therefore provides evidence that
genetically engineered mice can be used to model human lung disease resulting
from Rb deficiency.
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ACKNOWLEDGMENTS |
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