THE ANATOMICAL RECORD 263:25–34 (2001)
Functional Overexpression of WildType p53 Correlates With Alveolar
Cell Differentiation in the Developing
Human Lung
MARIA TEBAR,* JOOST J.M. BOEX, AND ANK A.W. TEN HAVE-OPBROEK
Department of Pulmonology, Leiden University Medical Center, Leiden,
The Netherlands
ABSTRACT
At 15 weeks after conception (a.c.), the human pulmonary acinus is
lined by distal low-columnar and more proximal cuboidal cells that are
successive stages in alveolar type II cell differentiation (pseudoglandular
period of lung development). From 16 weeks a.c. onward, there are also
’flatter’ cells that are intermediate stages in the differentiation of cuboidal
type II cells into squamous type I cells (canalicular period). We investigated
the role of wild-type p53 protein and the proliferation marker Ki-67 in the
differentiation of type II and type I cells in these two periods. Serial sections
from fetal lungs (n ⫽ 30) were immunoincubated with antibodies against
p53 and Ki-67. The presence of prospective type II and type I cells was
confirmed using immunohistochemistry for surfactant protein SP-A as a
differentiation marker and light and electron microscopy. The p53 and
Ki-67 positive nuclei were quantified per alveolar cell phenotype (i.e., lowcolumnar; cuboidal; flatter). The occurrence of cell apoptosis was studied
using propidium iodide (PI) and 4⬘,6⬘-diamino-2-phenylindol dihydrochloride (DAPI) staining. The combined increase in p53 expression and decrease
in Ki-67 expression during alveolar epithelial cell differentiation suggests
that wild-type p53 protein plays a role in the differentiation of alveolar type
II and type I cells in the human lung, and that this function is mediated
through cell cycle arrest. The rare incidence of apoptotic nuclei in alveolar
type II cells, together with their absence in alveolar type I cells, supports
the view that p53 is involved in the differentiation, rather than the death,
of alveolar epithelial cells. Anat Rec 263:25–34, 2001.
©
2001 Wiley-Liss, Inc.
Key words: p53; Ki-67; alveolar type I cells; alveolar type II cells;
cell differentiation; cell cycle arrest
The primitive pulmonary acinus is detectable in human
fetal lungs from 10 –12 weeks after conception (a.c.) onward. At that time, it is composed of acinar tubules lined
by prospective alveolar type II cells (Otto-Verberne et al.,
1988). These early prenatal type II cells show a large and
round nucleus, large apical and basal glycogen fields, a
rough and smooth endoplasmic reticulum, a well-developed Golgi apparatus with many associated vesicles and
various types of inclusion bodies (cytoplasmic, granular/
flocculent, osmiophilic, multivesicular and dense), claimed
to be precursory stages of multilamellar bodies (Ten HaveOpbroek, 1991; Ten Have-Opbroek et al., 1988, 1990b,
1991). On the basis of these ultrastructural features, al©
2001 WILEY-LISS, INC.
veolar type II cells can be easily recognized from bronchiolar Clara cells, the other major secretory cell type in the
peripheral lung (Plopper et al., 1980; Ten Have-Opbroek
and De Vries, 1993; Plopper and Ten Have-Opbroek,
1994). In this so-called pseudoglandular period of lung
*Correspondence to: Dr. Maria Tebar, Respiratory Biology
Group, c/o Anatomy, PO Box 9602, NL-2300 RC Leiden, The
Netherlands. E-mail: [email protected]
Received 7 September 2000; Accepted 15 November 2000
Published online 00 Month 2001
26
TEBAR ET AL.
development, two stages in the differentiation of prospective type II cells can be distinguished: a low-columnar cell,
located in the end-pieces of the acinus and having a high
glycogen content, and a cuboidal cell situated more proximally and with a low glycogen content. Around 16 weeks
a.c., there is also a third stage in the alveolar epithelial
cell differentiation, mainly in those areas of the acinus
that are close to capillaries. This is a ’flatter’ cell type,
ranging in shape from cuboidal to squamous, that represents an intermediate stage in the differentiation of prospective cuboidal type II cells into prospective squamous
type I cells (Ten Have-Opbroek, 1979, 1991; Otto-Verberne and Ten Have-Opbroek, 1987; Otto-Verberne et al.,
1988; Ten Have-Opbroek and Plopper, 1992; Brandsma et
al., 1993, 1994). As a consequence of this cell ‘flattening,’
the acinar tubules start to transform into derivative structures, namely prospective alveolar ducts and sacs (canalicular period of lung development). Our group has been
the first to demonstrate that, in human fetal lungs, prospective type II and type I cells display specific protein
expression, notably surfactant protein SP-A, as soon as
they appear in the pseudoglandular period of lung development (Otto-Verberne et al., 1988, 1990). This has also
been shown for the mouse (Ten Have-Opbroek, 1975,
1979), the rat (Otto-Verberne and Ten Have-Opbroek,
1987) and the Rhesus monkey (Ten Have-Opbroek and
Plopper, 1992). These results have been confirmed by
other groups (reviewed in Ten Have-Opbroek and Plopper,
1992).
The p53 tumor suppressor gene encodes a 53 kD protein
that is an important key in the regulation of the normal
cell cycle. The wild-type p53 protein is active as a transcription factor and able to initiate cell cycle arrest at the
G0/G1 checkpoint (Dulic et al., 1994; Mercer et al., 1990;
Yin et al., 1992). Recent in vitro and in vivo studies indicate that the process for cells to undergo differentiation
involves up-regulation of the p53 expression (Rotter et al.,
1994; Aloni-Grinstein et al., 1995; Eizenberg et al., 1996;
Almog and Rotter, 1997). These data, together with the
finding that p53 mRNA and protein are expressed during
mouse embryogenesis, with high levels of expression in
specific differentiating tissues (Louis et al., 1988; Schmid
et al., 1991), suggest that p53 up-regulation plays a role in
organogenesis.
Therefore, the aim of this study in the early fetal human
lung (15–20 weeks a.c.) was to investigate the expression
of wild-type p53 protein in relation to the expression of a
cell proliferation marker (Ki-67) during alveolar cell differentiation. For this, we used immunohistochemistry in
serial sections and morphometric analysis. Prospective
alveolar cells were identified using an antibody against
SP-A and light and electron microscopy. In addition, two
apoptosis stainings were used to examine the occurrence
of cell death. Our results are consistent with the possibility that wild-type p53 regulates alveolar type II and type
I cell differentiation during human lung development
through cell cycle arrest.
MATERIALS AND METHODS
Permission to use human fetal tissue specimens was
given by the Committee for Medical Ethics of the Medical
School of the University of Leiden.
TABLE 1. Developmental ages of human fetuses
included in Group I (pseudoglandular period of lung
development) and Group II (canalicular period of
lung development) in the present study
Developmental agea
Group I
15
15 ⫹ 0 (n ⫽ 6)
15 ⫹ 2 (n ⫽ 1)
16 ⫹ 0 (n ⫽ 8)
16
17
18
19
20
Group II
16 ⫹ 4 (n ⫽ 1)
16 ⫹ 5 (n ⫽ 1)
17 ⫹ 0 (n ⫽ 1)
17 ⫹ 2 (n ⫽ 1)
17 ⫹ 5 (n ⫽ 1)
18 ⫹ 0 (n ⫽ 4)
18 ⫹ 3 (n ⫽ 1)
19 ⫹ 0 (n ⫽ 1)
19 ⫹ 2 (n ⫽ 1)
19 ⫹ 3 (n ⫽ 1)
19 ⫹ 5 (n ⫽ 1)
20 ⫹ 0 (n ⫽ 1)
a
Expressed in weeks and additional days after conception.
The developmental age was estimated by measuring the distantia biparietalis (head diameter) using ultrasound. When
the fetus was younger than 18 weeks a.c., the foot length was
included.
Tissue Preparation
The study was carried out on lungs from 30 human
fetuses (aged 15–20 weeks a.c.), obtained from the “Medical Center for Birth Control” in Leiden. Curettage material was placed in ice-cold phosphate buffered saline (PBS,
pH 7.3) immediately after abortion. Macroscopically identifiable lung lobes were transferred to 4% buffered paraformaldehyde (pH 7.3) and fixed by immersion for 16 hr at
room temperature (rt). After dehydration through a
graded ethanol series (70 –100%, each step lasting 30
min), the tissue was embedded in paraffin and serially
sectioned at 5 ␮m. The sections were mounted on precoated slides (Knittel Gläser, Germany) and stored at rt.
Light Microscopy
Staining with hematoxylin and eosin (H&E) was performed on serial sections of each specimen to select blocks
for immunohistochemistry and for general orientation.
Based upon the presence of only prospective type II cells or
both prospective type II and type I cells in the lining of the
pulmonary acinus, specimens were divided in Group I
(pseudoglandular period) and Group II (canalicular period), respectively (Table 1).
Apoptosis Labeling
Using the technique of Coles et al. (1993), one series of
sections was incubated with 4 ␮g/ml PI (propidium iodide;
Serva Feinbiochemica, Heidelberg, Germany) and 100
␮g/ml RNase (Sigma, St. Louis, MO) in PBS for 30 min at
rt. A second series of sections was stained with 0.1% DAPI
(4⬘,6⬘-diamino-2-phenylindol dihydrochloride; Boehringer
Mannheim, Germany) in aqua dest for 15 min at rt (Salido
et al., 2000). All sections were mounted with antifading
mounting medium for fluorescence (500␮g/ml ␳-phenylenediamine, Sigma, and 38% glycerol, Merck, Darmstadt,
Germany, in PBS) and stored in darkness at ⫺20°C until
examination.
27
P53 AND ALVEOLAR CELL DIFFERENTIATION
Antibodies
For type II cell detection, we used a polyclonal antibody
against surfactant protein A (SP-A) called SALS-Hu
(Otto-Verberne et al., 1988, 1990). SALS-Hu was prepared
in our laboratory by injecting rabbits with the pellet fraction of bronchoalveolar lavage fluid from adult human
lung. The resulting immune serum was absorbed with
serum and cross-reacting organs (Otto-Verberne et al.,
1988). The SP-A specificity of the final immune serum was
assessed by western blot analysis, in vitro translation
assays of mRNA from human lungs, immunoprecipitation
and immunohistochemistry. SALS-Hu recognizes recombinant human SP-A in western blots and can be depleted
with this substance (Otto-Verberne et al., 1988, 1990). The
antibody was used in our further studies of lung morphogenesis (Otto-Verberne et al., 1991; Ten Have-Opbroek et
al., 1991; Ten Have-Opbroek and Plopper, 1992) and
pathogenesis (Ten Have-Opbroek et al., 1990a, 1997).
In addition, we used a monoclonal antibody against the
DNA replication control protein p53 (Clone DO-1, mouse
anti-human; Santa Cruz Biotechnology, Santa Cruz, CA),
and a monoclonal antibody against the cell proliferation
marker Ki-67 (Clone MIB-1, mouse anti-human; Immunotech, Marseille, France). Ki-67 is a nuclear antigen
present in all proliferating cells that are in active phases
of the cell cycle, i.e., G1, S, G2 and mitosis, but which is
absent in G0 cells, and therefore it can be used as a
proliferation marker (Gerdes, 1990).
Immunohistochemistry
Immunohistochemical staining was performed using
the avidin-biotin complex (ABC) method with peroxidase
labeling, and 3-3⬘diaminobenzidine (DAB) as the chromogen (Vector; Burlingame, CA). Briefly, the procedure involves the following steps: 1) hydration of the paraffin
sections through xylene and a graded ethanol series
(100 —70%, each step lasting 30 min) and quenching of the
endogenous peroxidase activity with 100% methanol containing 0.4% hydrogen peroxide (H2O2) for 20 min at rt; 2)
three times rinsing in Tris Maleate buffer (TMB, pH 7.6)
for 1 min at rt and incubation with 10% normal horse
serum for 1 hr at rt; 3) incubation with the primary antibody (SALS-Hu, 1:50; anti-p53, 1:2,000; anti-Ki-67, 1:100;
all diluted in TMB, pH 7.6) overnight at 4°C and rinsing in
TMB; 4) incubation with a 1:400 dilution of biotinylated
swine anti-rabbit IgG (Dako, Denmark) (after SALS-Hu)
or biotinylated horse anti-mouse IgG (after anti-p53 or
anti-Ki-67) for 60 min at rt and rinsing in TMB; 5) incubation with ABC for 30 min at rt and rinsing in TMB; and
6) incubation with TMB containing 0.04% DAB and
0.006% H2O2 for 10 min at rt. Finally, the sections were
washed in TMB for 1 min and in tap water for 10 min,
then counterstained for 5 sec with hematoxylin that
stained the various nuclei bluish, rinsed in tap water for
10 min, dehydrated through a graded ethanol series (70 –
100%) and xylene and mounted with xylene-soluble
mounting medium Depex (H.D. Supplies, England).
For the detection of wild-type p53 protein, we used an
enhancement procedure, i.e., deposition of biotinylated
tyramide (Kerstens et al., 1995). After incubation with
ABC (Step 5), the sections were incubated with 1:400
biotinylated tyramide in TMB containing 0.006% H2O2 for
10 min at rt, then rinsed in TMB, and again incubated
with ABC before continuing with Step 6.
Immunohistochemical controls were performed on the
serial human fetal lung sections using preimmunization
serum or normal mouse serum as the primary antibody, or
omission of one of the incubation steps.
Morphometry
In sections immunoincubated with anti-p53 or antiKi-67 antibodies, microscopic fields were chosen by random serial sampling (Gundersen and Jensen, 1987). We
took 10 sections per specimen with an interval of 50 ␮m.
The number of p53 and Ki-67 positive nuclei were counted
per alveolar cell type (i.e., low-columnar, cuboidal and
flatter cells). For each cell type, 100 or more nuclei were
counted over at least 10 microscopic fields.
Statistical Analysis
The morphometric data are expressed as mean ⫾ SEM.
Quantitative data of the p53 and Ki-67 positive nuclei
were analyzed statistically by using the one-way analysis
of variance (ANOVA) followed by the Student-NewmanKeuls’ Multiple Range Test in two ways: a) per alveolar
cell type, and b) per protein expression; a difference was
considered to be statistically significant if P ⬍ 0.05.
Electron Microscopy
Lung lobes were fixed by immersion in 2% paraformaldehyde and 1.25% glutaraldehyde in 0.1 M cacodylate
buffer (pH 7.4) for 24 hr at 4°C and postfixed in 1%
osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4) for 2
hr at rt. After dehydration through a graded ethanol series (70 –100%), the tissue was processed for transmission
(TEM) and scanning electron microscopy (SEM) as described previously (Ten Have-Opbroek et al., 1988).
RESULTS
Light Microscopy
In the pseudoglandular period of lung development, the
epithelial lining displayed two stages in alveolar cell differentiation, which differed in morphology and location:
the low-columnar cells were located in the end-pieces of
the acini, whereas the cuboidal cells were situated more
proximally. Large areas that did not stain with eosin could
be recognized in the basal and apical cytoplasm. As reported (Otto-Verberne et al., 1988) and shown below
through electron microscopy, these areas corresponded to
glycogen fields. Such fields were especially visible in the
distal low-columnar shaped cells. At this early fetal age,
only some capillaries were present underneath the alveolar epithelial layer. Similar to the period before, the epithelial lining in the canalicular period of lung development exhibited distal low-columnar and proximal cuboidal
cells with a similar distribution (see above). Additionally,
there was a third stage in alveolar cell differentiation,
namely cells ranging in shape from cuboidal to squamous,
which have been indicated as ’flatter’ cells (Ten HaveOpbroek, 1979, 1991). These flatter cells were also found
more proximally in close relationship with capillaries that
were more abundant in this period.
Electron Microscopy
Figure 1A is a representative scanning electron micrograph of the tubular system in the early fetal human lung.
It shows the transition from the pseudostratified colum-
28
TEBAR ET AL.
Fig. 1. Scanning (SEM) and transmission (TEM) electron microscopy
of the pulmonary acinus in human fetuses. A: Eighteen weeks a.c. (SEM).
Note the transition from the pseudostratified columnar epithelium of the
prospective bronchial portion (BR) to the cuboidal epithelium of the
prospective respiratory portion (pulmonary acinus, PA) of the lung. SA:
saccular ending (⫻320). B: Fifteen weeks a.c. TEM micrograph of the
prospective pulmonary acinus showing low-columnar and approximately cuboidal cells with large round nuclei. Glycogen fields (GL) are
located in the apical and basal cytoplasm. Mitochondria, endoplasmic
reticulum and cytoplasmic inclusions are mostly present close to the
apical cell border. BL: basal lamina (⫻4,370). C: As of 16 weeks a.c.
(TEM), the epithelial lining also contains shorter cells (arrowhead) situated above capillaries (CA) (⫻4,370). D: Eighteen weeks a.c. (TEM). An
attenuated cell (arrowhead) is present adjacent to a capillary (CA)
(⫻3,200).
nar epithelium of the prospective bronchial portion to the
approximately cuboidal epithelium of the prospective respiratory portion (unit: pulmonary acinus) of the lung.
Figures 1B–D are transmission electron micrographs of
the epithelial cells lining the acinar tubules of fetuses in
Group I (Fig. 1B) and in Group II (Fig. 1C,D). In the
pseudoglandular period of lung development, there were
low-columnar and approximately cuboidal cells with large
and more or less round nuclei, showing blunt microvilli
and cellular protrusions at the luminal side, a basal lam-
ina, and interdigitations and junctional complexes with
adjacent epithelial cells. Large glycogen fields were located in the apical and basal parts of the cells. Cell organelles such as mitochondria, smooth and rough endoplasmic reticulum and varying types of inclusion bodies
(cytoplasmic, granular/flocculent, multivesicular and
dense) predominated in the cytoplasmic area close to the
apical plasma membrane. Compartments formed by
smooth membrane systems were regularly seen in the
glycogen fields (Fig. 1B). In the canalicular period of lung
P53 AND ALVEOLAR CELL DIFFERENTIATION
Fig. 2. Immunohistochemistry of the pulmonary acinus in human
fetuses (incubated with SP-A antibody). A: Sixteen weeks a.c. The
low-columnar and cuboidal epithelial cells located in the distal (asterisk)
and in the more proximal (square) regions of the acini, respectively, show
positive (⫽ yellow to red) cytoplasmic staining for SP-A, especially in the
apical area (⫻132). B: Detail of A, exhibiting the cytoplasmic SP-A
staining. Note the absence of staining in the glycogen fields around the
large and roundish nuclei (⫽ bluish) (⫻330). C: At 19 weeks a.c., a strong
apical staining for SP-A is seen in both cuboidal and flatter (FL) cells.
Nuclear counterstaining: hematoxylin (⫻330).
29
Fig. 3. Immunohistochemistry of the pulmonary acinus in human
fetuses (incubated with p53 antibody). A: Sixteen weeks a.c. The lowcolumnar cells present in the distal regions (asterisk) of the acini show
less p53 positive (⫽ brown) nuclear staining than the cuboidal (CU) cells
present in more proximal regions of the acini (⫻132). B: Nineteen weeks
a.c. The cuboidal cells display the same p53 staining pattern as seen at
16 weeks a.c. (see A). In addition, almost all the flatter (FL) cells are
positive for p53 (⫻132). C: Detail of B, showing the intense p53 immunostaining in flatter cells. Nuclear counterstaining: hematoxylin (⫽ bluish)
(⫻330).
30
TEBAR ET AL.
Fig. 7. Apoptosis staining of the pulmonary acinus in human fetuses
(incubated with DAPI). Representative image of acinar tubules in a 19
weeks a.c. specimen. Two nuclei of mesenchymal cells near the epithelium appear brighter and smaller or fragmented, showing two different
stages in the apoptotic process (arrows). The nuclei of alveolar cells
lining the acinar tubules do not show any sign of apoptosis. A: Acinar
tubule. B: Bronchiole (⫻565).
development, the epithelial cells lining the acinar tubules
exhibited the same ultrastructural characteristics as described above. Capillaries were seen located in the proximity of mildly (Fig. 1C) and strongly (Fig. 1D) attenuated
cuboidal cells.
Immunohistochemistry
Fig. 4. Immunohistochemistry of the pulmonary acinus in human
fetuses (incubated with Ki-67 antibody). A: Sixteen weeks a.c. Strong
Ki-67 positive (⫽ brown) nuclear staining locates to the low-columnar
cells present in the distal regions (asterisk) of the acini; the cuboidal cells
present in more proximal regions (square) of the acini show less Ki-67
immunostaining (⫻132). B: Nineteen weeks a.c. The cuboidal (CU) cells
display the same Ki-67 staining pattern as found at 16 weeks a.c. (see A),
whereas the nuclei of the flatter (FL) cells are Ki-67 negative (⫻132). C:
detail of B. Nuclear counterstaining: hematoxylin (⫽ bluish) (⫻330).
After incubation with the anti-SP-A antibody (SALSHu), the sections of fetal lungs in Group I showed positive
(⫽ yellow to red) staining in the epithelial lining composed
of distal low-columnar and proximal cuboidal cells (Fig.
2A). The SP-A reactivity was present in the cytoplasm,
especially in the apical areas and along the cell borders.
The large glycogen fields around the nucleus (⫽ bluish)
often remained unstained (Fig. 2B). In the sections of fetal
lungs in Group II, the distal low-columnar and proximal
cuboidal cells showed a staining pattern for SP-A similar
to the one described above. Additionally, SP-A reactivity
was also found in the proximal flatter cells (Fig. 2C).
P53 AND ALVEOLAR CELL DIFFERENTIATION
31
Fig. 8. Diagram showing the spatial distribution of the various nuclear markers used in the present study in a representative fetal acinus
in Group II. Ki-67 positive nuclei predominate in the end-pieces of the
acinus, lined by low-columnar (i.e., immature) alveolar type II cells. By
contrast, p53 positive nuclei prevail in more proximal regions of the
acinus, lined by cuboidal (i.e., more mature) alveolar type II cells and
flatter (i.e., immature) alveolar type I cells. Occasionally, immature alveolar type II cells show an apoptotic nucleus. For further explanation, see
Results.
After incubation with the anti-p53 antibody, the sections of fetal lungs in both Group I and Group II demonstrated positive (⫽ brown) nuclear staining in the distal
low-columnar and the proximal cuboidal cells (Fig. 3A). In
Group II, p53 positive nuclei were more abundant in the
proximal flatter cells than in the other alveolar cell types
(Fig. 3B,C).
The sections of fetal lungs in Group I immunoincubated
with the anti-Ki-67 antibody exhibited positive (⫽ brown)
nuclear staining in the two alveolar cell types present, i.e.,
distal low-columnar and proximal cuboidal cells, but particularly in the former one (Fig. 4A). In the sections of fetal
lungs in Group II, the nuclear reactivity for Ki-67 protein
was positive and comparable in both distal low-columnar
and proximal cuboidal cells, whereas the proximal flatter
cells were almost negative for Ki-67 expression (Fig. 4B,C).
cells compared to the distal low-columnar cells (P ⬍ 0.05)
(Fig. 5). Remarkably, when comparing the expression of
both proteins, there were more p53 than Ki-67 positive
nuclei in the proximal cuboidal cells (P ⬍ 0.05).
The quantification of the p53 and Ki-67 expression data
in Group II indicated that there was no significant difference between the distal low-columnar and the proximal
cuboidal cells for the expression of both proteins. By contrast, the percentage of p53 positive nuclei was significantly higher in the proximal flatter cells compared to the
other two alveolar cell types (P ⬍ 0.001) (Fig. 6). Likewise,
the percentage of Ki-67 positive nuclei was significantly
lower in the proximal flatter cells when compared with the
distal low-columnar cells (P ⬍ 0.001) and the proximal
cuboidal cells (P ⬍ 0.01) (Fig. 6). Finally, when comparing
the expression of p53 and Ki-67, we found an inverse
relationship between them during the progression of alveolar cell differentiation, being most prominent in the proximal flatter cells (P ⬍ 0.001).
Quantification of p53 and Ki-67 Expression
In sections of fetal lungs in Group I, the counting of the
p53 positive nuclei showed no significant difference between the distal low-columnar and the proximal cuboidal
cells. The percentage of the Ki-67 positive nuclei was
significantly smaller, however, in the proximal cuboidal
Detection of Apoptosis
Under the fluorescence microscope, normal alveolar cell
nuclei in human fetal lungs in groups I and II displayed a
32
TEBAR ET AL.
ptotic nuclei were found in the end-pieces of the acini,
where they occurred in distal low-columnar (and occasionally proximal cuboidal) cells, whereas no sign of apoptosis
was detected in the nuclei of proximal flatter cells.
The spatial distribution of the various nuclear markers
used in this study, in a representative acinus in Group II,
is shown in a diagram (Fig. 8). Although most of the
low-columnar prospective alveolar type II cells, located to
the distal ends of the pulmonary acinus, are positive for
the proliferation marker Ki-67, the cuboidal prospective
type II cells present more proximally show both Ki-67 and
p53 positive nuclei. The proximal flatter prospective type
I cells display nuclear immunoreactivity for p53 only. On
the other hand, apoptotic nuclei are found infrequently in
the interstitium close to the epithelial lining as well as
occasionally in distal low-columnar prospective type II
cells. A similar spatial distribution of the nuclear markers
in the acini was found in Group I.
Fig. 5. Histogram showing the percentage of p53 and Ki-67 positive
nuclei per alveolar cell type in human fetal lungs in Group I (pseudoglandular period). Each column indicates the mean value obtained from 15
specimens ⫾ SEM. a: significant difference (P ⬍ 0.05) for Ki-67 expression between the distal low-columnar cells and the proximal cuboidal
cells; b: significant difference (P ⬍ 0.05) in the proximal cuboidal cells
between p53 and Ki-67 expression (one-way ANOVA followed by Student-Newman-Keuls’ Multiple Range test).
Fig. 6. Histogram showing the percentage of p53 and Ki-67 positive
nuclei per alveolar cell type in human fetal lungs in Group II (canalicular
period). Each column indicates the mean value obtained from 15 specimens ⫾ SEM. a: significant difference (P ⬍ 0.001) for p53 expression
between the proximal flatter cells and the other two alveolar cell types.
b: significant difference for Ki-67 expression between the proximal flatter
cells and the proximal cuboidal cells (P ⬍ 0.01) and between the proximal flatter cells and the distal low-columnar cells (P ⬍ 0.001). c: significant difference (P ⬍ 0.001) in the proximal flatter cells between p53 and
Ki-67 expression (one-way ANOVA followed by Student-Newman-Keuls’
Multiple Range test).
more or less round shape and a red (PI) or blue (DAPI)
staining with one or two nucleoli. By contrast, apoptotic
nuclei were smaller because of shrinkage and more
brightly stained than normal nuclei. They were often fragmented and showed marked condensation of chromatin
(Fig. 7). In both age groups studied, apoptosis was rare
and located mainly to the mesenchymal compartment,
close to the epithelial lining. Infrequently, disperse apo-
DISCUSSION
Functional Overexpression of p53 Protein in
Prospective Alveolar Type II and Type I Cells
The aim of the present work was to investigate the role
of p53 and Ki-67 in the differentiation of prospective alveolar type II and type I cells in the late pseudoglandular
and early canalicular periods of lung development. Our
present data are consistent with the possibility that, at
this specific time of lung development, one alveolar cell
lineage with different morphologic phenotypes and locations lines the components of the primitive pulmonary
acinus [called acinar tubules (Ten Have-Opbroek, 1979)].
The distal low-columnar and the proximal cuboidal alveolar cells in the epithelial lining exhibited the distinctive
ultrastructural features of prospective alveolar type II
cells (see Introduction) and showed positive cytoplasmic
staining for SP-A. The third, also proximal, alveolar cell
type that appeared in the early canalicular period of lung
development, was often located in close proximity to capillaries. As presently shown, these cells were always SP-A
positive, irrespective of their varying shapes (cuboidal to
almost squamous). These so-called ’flatter’ cells (Ten
Have-Opbroek, 1979, 1991) are considered to be intermediate stages in the differentiation of cuboidal type II into
squamous type I cells. Fully differentiated squamous alveolar type I cells as seen in the adult lung are always
SP-A negative (Ten Have-Opbroek et al., 1991). All these
observations agree with, and also extend, previous reports
on the presence of prospective alveolar type II and type I
cells in developing lungs in the human as well as in other
species (reviewed in Ten Have-Opbroek and Plopper,
1992).
The present study detects positive reactivity for p53
protein in the pulmonary acinus of the human fetal lung
from 15–20 weeks a.c. The p53 nuclear staining is present
in all three stages of alveolar epithelial cell differentiation
(i.e., distal low-columnar, proximal cuboidal and proximal
flatter cells). Expression of wild-type p53 protein in the
cell nuclei is a normal physiological response to slow down
the cell cycle at the G1 phase. This protein is not usually
detectable by immunohistochemistry because it is shortlived in comparison with its mutated counterpart and
present at low levels. It may become detectable, however,
by immunohistochemical means in normal tissues if accumulated due to functional overexpression (Battifora,
P53 AND ALVEOLAR CELL DIFFERENTIATION
1994). The enhancement procedure used in the present
experiments allowed us to detect nuclear reactivity for p53
in a specific expression pattern in prospective alveolar
type II and type I cells. This finding suggests that p53
protein is functionally overexpressed during alveolar cell
differentiation (see below). Interestingly, our results are
in agreement with other studies reporting that wild-type
p53 is found in the nuclei of differentiating normal and
tumor cells (Moll et al., 1995; Eizenberg et al., 1996).
Consequently, all these data are compatible with the idea
(Eizenberg et al., 1996; Almog and Rotter, 1997) that p53
nuclear localization and transcriptional activity is necessary for cellular differentiation.
As for the proliferation marker Ki-67, we found nuclear
staining in the lining of the fetal human pulmonary acinus
from 15–20 weeks a.c. This finding is in accordance with
the fact that this antigen is present in the nuclei of cells
that are in active parts of the cell cycle (Gerdes, 1990).
Correlation of p53 Functional Overexpression
to Alveolar Cell Differentiation
The quantification of p53 and Ki-67 expression in prospective alveolar type II cells (i.e., distal low-columnar
and proximal cuboidal cells) in the pseudoglandular period of lung development showed that the percentage of
wild-type p53 positive nuclei was higher in proximal
cuboidal cells in comparison with distal low-columnar
cells, although the difference was not statistically significant. At the same time, proximal cuboidal cells exhibited a
lower percentage of Ki-67 positive nuclei compared to
distal low-columnar cells. Because the wild-type p53 protein has been shown to have an antiproliferative effect by
blocking cell cycle progression (Mercer et al., 1990) and to
selectively down-regulate the expression of the proliferating cell nuclear antigen (Mercer et al., 1991), we assume
that the decrease in the proliferation rate (as measured by
Ki-67 expression) of proximal cuboidal cells in relation to
distal low-columnar cells is the result of a p53-induced
growth arrest. A similar mechanism seems to be involved
in type I cell differentiation. For in the canalicular period
of lung development, we observed the same events as
described above together with a remarkable inverse correlation between p53 and Ki-67 expression in the proximal
flatter cells. In summary, the finding of rising p53 expression levels synchronically with declining Ki-67 expression
levels during alveolar epithelial cell attenuation (from
low-columnar to cuboidal and from this to flatter) strongly
suggests that the wild-type p53 protein participates in
alveolar type II and type I cell differentiation in the developing human lung, probably through cell cycle arrest.
CONCLUSIONS
Our conclusion that p53 plays a role in alveolar cell
differentiation is in line with other reports in the literature. Lutzker and Levine (1996) have shown that induction of cell differentiation in undifferentiated teratocarcinoma cell lines enhances p53 half life and p53 target genes
transcriptional activity. Embryonic studies indicate that
the wild-type p53 protein may play an important role as a
positive regulator of cell differentiation (Halevy, 1993;
Woodworth et al., 1993; Keren-Tal et al., 1995; Eizenberg
et al., 1996; Almog and Rotter, 1997). Likewise, other
tumor suppressor genes have been found to be essential
for embryonic development in the mouse (for review, see
33
Jacks, 1996). Throughout mouse embryogenesis, a strong
p53 signal is expressed during specific differentiation
stages of certain tissues like brain, lung, thymus, intestine, salivary gland and kidney (Schmid et al., 1991). This
participation of the wild-type p53 protein in mouse embryogenesis seems to be in contradiction with the first
studies (Donehower et al., 1992) carried out on p53 knockout mice because the latter describe an apparent normal
prenatal and postnatal development. One possible explanation for this discrepancy might be that the function of
p53 in cell differentiation is masked by other compensating genes or alternative pathways in mammalian species
as has been proposed by Almog and Rotter (1997) and Choi
and Donehower (1999). Later findings have reported that
p53 null mice show, at certain frequencies, developmental
alterations (Armstrong et al., 1995; Sah et al., 1995). Furthermore, the fact that transgenic mice overexpressing
mutant alleles of the p53 protein show a high incidence of
lung adenocarcinomas (Lavigueur et al., 1989), confirms
the direct role of this protein as a negative regulator of cell
proliferation in the epithelial compartment of the lung.
Because wild-type p53 is also implicated in the pathway
inducing apoptosis (Yonish-Rouach et al., 1991, 1993;
Oren, 1994), it could be that the p53 overexpression in the
fetal lung reported in the present experiments is related to
the induction of cell death rather than cell maturation.
The present study did not detect any distinct sign of nuclear fragmentation in differentiating alveolar type I cells,
i.e., proximal flatter cells, and only sporadic apoptotic
nuclei in distal low-columnar and proximal cuboidal alveolar type II cells. In agreement with our observations, it
has been reported that all apoptotic cells found in human
fetal lungs aged 15–24 weeks a.c. were located in the
interstitium, whereas in epithelial cells no apoptotic nuclei were detected (Scavo et al., 1998). This finding further
supports our present concept of p53 acting exclusively as a
regulator of cell differentiation in alveolar epithelial cells
of the early fetal human lung.
In conclusion, our results indicate that the wild-type
p53 protein may play a role in alveolar epithelial cell
differentiation in the developing human lung by interfering with the normal cell cycle progression. More research
is needed to achieve further insight into the mechanism
through which wild-type p53 regulates normal cell proliferation and differentiation, as well as its role in developmental biology, carcinogenesis and wound repair.
LITERATURE CITED
Almog N, Rotter V. 1997. Involvement of p53 in cell differentiation
and development. Biochim Biophys Acta 1333:F1–F27.
Aloni-Grinstein R, Schwartz D, Rotter V. 1995. Accumulation of wildtype p53 protein upon gamma-irradiation induces a G2-dependent
immunoglobulin k light chain gene expression. EMBO J 14:1392–
1401.
Armstrong JF, Kaufman MH, Harrison DJ, Clarke AR. 1995. Highfrequency developmental abnormalities in p53-deficient mice. Curr
Biol 5:931–936.
Battifora H. 1994. P53 Immunohistochemistry: a word of caution.
Hum Pathol 25:435– 436.
Brandsma AE, Tibboel D, Vulto IM, Egberts J, Ten Have-Opbroek
AAW. 1993. Ultrastructural features of alveolar epithelial cells in
the late fetal pulmonary acinus: a comparison between normal and
hypoplastic lungs using a rat model of pulmonary hypoplasia and
congenital diaphragmatic hernia. Microsc Res Techniq 26:389 –399.
Brandsma AE, W. Ten Have-Opbroek AAW, Vulto IM, Molenaar JC,
Tibboel D. 1994. Alveolar epithelial composition and architecture of
the late fetal pulmonary acinus: an immunocytochemical and mor-
34
TEBAR ET AL.
phometric study in a rat model of pulmonary hypoplasia and congenital diaphragmatic hernia. Exp Lung Res 20:491–515.
Choi J, Donehower LA. 1999. P53 in embryonic development: maintaining a fine balance. Cell Mol Life Sci 55:38-47.
Coles HSR, Burne JF, Raff MC. 1993. Large-scale normal cell death in
the developing rat kidney and its reduction by epidermal growth
factor. Development 118:777–784.
Donehower LA, Harvey M, Slagle BT, McArthur MJ, Montgomery CA,
Butel JS, Bradly A. 1992. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumors. Nature 356:
215–221.
Dulic V, Kaufmann WK, Wilson SJ, Tisty TD, Lees E, Harper JW,
Elledge SJ, Reed SI. 1994. P53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation
induced G1 arrest. Cell 76:1013–1023.
Eizenberg O, Faber-Elman A, Gottlieb E, Oren M, Rotter V, Schwartz
M. 1996. P53 plays a regulatory role in differentiation and apoptosis
of central nervous system-associated cells. Mol Cell Biol 16:5178 –
5185.
Gerdes J. 1990. Ki-67 and other proliferation markers useful for
immunohistological diagnostic and prognostic evaluations in human malignancies. Semin Cancer Biol 1:199 –206.
Gundersen HJG, Jensen EB. 1987. The efficiency of systematic sampling in stereology and its prediction. J Micros Oxford 147:229 –263.
Halevy O. 1993. P53 gene is up-regulated during skeletal muscle cell
differentiation. Biochem Biophys Res Comm 192:714 –719.
Jacks T. 1996. Tumor suppressor gene mutations in mice. Annu Rev
Genet 30:603– 636.
Keren-Tal I, Suh B, Dantes A, Linder S, Oren M, Amsterdam A. 1995.
Involvement of p53 expression in cAMP-mediated apoptosis in immortalized granulosa cells. Exp Cell Res 218:283–295.
Kerstens HMJ, Poddighe PJ, Hanselaar GJM. 1995. A novel in situ
hybridization signal amplification method based on the deposition
of biotinylated tyramide. J Histochem Cytochem 43:347–352.
Lavigueur A, Maltby V, Mock D, Rossant J, Pawson T, Bernstein A.
1989. High incidence of lung, bone and lymphoid tumors in transgenic mice overexpressing mutant alleles of the p53 oncogene. Mol
Cell Biol 9:3982–3991.
Louis JM, McFarland VW, May P, Mora PT. 1988. The phosphoprotein p53 is down-regulated post-transcriptionally during embryogenesis in vertebrates. Biochem Biophys Acta 950:395– 402.
Lutzker SG, Levine AJ. 1996. A functionally inactive p53 protein in
teratocarcinoma cells is activated by either DNA damage or cellular
differentiation. Nat Med 2:804 – 810.
Mercer WE, Shields MT, Amin M, Sauve GJ, Appella E, Romano JW,
Ullrich SJ. 1990. Negative growth regulation in a glioblastoma
tumor cell line that conditionally expresses human wild-type p53.
Proc Natl Acad Sci USA 87:6166 – 6170.
Mercer WE, Shields MT, Lin D, Appella E, Ullrich SJ. 1991. Growth
suppression induced by wild-type p53 protein is accompanied by
selective down-regulation of proliferating-cell nuclear antigen expression. Proc Natl Acad Sci USA 88:1958 –1962.
Moll U, LaQuaglia M, Benard J, Rious G. 1995. Wild-type p53 protein
undergoes cytoplasmic sequestration in undifferentiated neuroblastomas but not in differentiated tumors. Proc Natl Acad Sci USA
92:4407– 4411.
Oren M. 1994. Relationship of p53 to the control of apoptotic cell
death. Semin Cancer Biol 5:221–227.
Otto-Verberne CJM, Ten Have-Opbroek AAW. 1987. Development of
the pulmonary acinus in fetal rat lung: a study based on an antiserum recognizing surfactant-associated proteins. Anat Embryol
175:365–373.
Otto-Verberne CJM, Ten Have-Opbroek AAW, Balkema JJ, Franken
C. 1988. Detection of the type II cell or its precursor before Week 20
of human gestation, using antibodies against surfactant-associated
proteins. Anat Embryol 178:29 –39.
Otto-Verberne CJM, Ten Have-Opbroek AAW, De Vries ECP. 1990.
Expression of the major surfactant-associated protein, SP-A, in type
II cells of human lung before 20 weeks gestation. Eur J Cell Biol
53:13–19.
Otto-Verberne CJM, Ten Have-Opbroek AAW, Willems LNA, Franken C, Kramps JA, Dijkman JH. 1991. Lack of type II cells and
emphysema in human lungs. Eur Respir J 4:316 –322.
Plopper CG, Hill LH, Mariassy AT. 1980. Ultrastructure of the nonciliated bronchiolar epithelial (Clara) cell of mammalian lung. III. A
study of man with comparison of 15 mammalian species. Ex Lung
Res 1:171–180.
Plopper CG, Ten Have-Opbroek AAW. 1994. Anatomical and histological classification of the bronchioles. In: Epler GR, editor. Diseases of the bronchioles. 1st edition. New York: Raven Press. p
15–25.
Rotter V, Aloni-Grinstein R, Schwartz D, Elkind NB, Simons A,
Wolkowicz R, Lavigne M, Besserman P, Kapon A, Goldfinger N.
1994. Does wild-type p53 play a role in normal cell differentiation?
Semin Cancer Biol 5:229 –236.
Sah VP, Attardi LD, Mulligan GJ, Williams BO, Bronson RT, Jacks T.
1995. A subset of p53-deficient embryos exhibit exencephaly. Nat
Genet 10:175–180.
Salido M, Vilches J, López A. 2000. Neuropeptides bombesin and
calcitonin induce resistance to etoposide induced apoptosis in prostate cancer cell lines. Histol Histopathol 15:729 –738.
Scavo LM, Ertsey R, Chapin CJ, Allen L, Kitterman JA. 1998. Apoptosis in the development of rat and human fetal lungs. Am J Respir
Cell Mol Biol 18:21–31.
Schmid P, Lorenz A, Hameister H, Montenarh M. 1991. Expression of
p53 during mouse embryogenesis. Development 113:857– 865.
Ten Have-Opbroek AAW. 1975. Immunological study of lung development in the mouse embryo. I. Appearance of a lung-specific antigen, localized in the great alveolar cell. Dev Biol 46:390 – 403.
Ten Have-Opbroek AAW. 1979. Immunological study of lung development in the mouse embryo. II. First appearance of the great
alveolar cell, as shown by immunofluorescence microscopy. Dev Biol
69:408 – 423.
Ten Have-Opbroek AAW. 1991. Lung development in the mouse embryo. Exp Lung Res 17:111–130.
Ten Have-Opbroek AAW, Dubbeldam JA, Otto-Verberne CJM. 1988.
Ultrastructural features of type II alveolar epithelial cells in early
embryonic mouse lung. Anat Rec 221:846 – 853.
Ten Have-Opbroek AAW, Hammond WG, Benfield JR. 1990a. Bronchioalveolar regions in adenocarcinoma arising from canine segmental bronchus. Cancer Lett 55:177–182.
Ten Have-Opbroek AAW, Otto-Verberne CJM, Dubbeldam JA. 1990b.
Ultrastructural characteristics of inclusion bodies of type II cells in
late embryonic mouse lung. Anat Embryol 181:317–323.
Ten Have-Opbroek AAW, Otto-Verberne CJM, Dubbeldam JA, Dijkman JH. 1991. The proximal border of the human respiratory unit,
as shown by scanning and transmission electron microscopy and
light microscopical cytochemistry. Anat Rec 229:339 –354.
Ten Have-Opbroek AAW, Plopper CG. 1992. Morphogenetic and functional activity of type II cells in early fetal rhesus monkey lungs. A
comparison between primates and rodents. Anat Rec 234:93–104.
Ten Have-Opbroek AAW, De Vries ECP. 1993. Clara cell differentiation in the mouse: ultrastructural morphology and cytochemistry
for surfactant protein A and Clara cell 10 kD protein. Microsc Res
Tech 26:400 – 411.
Ten Have-Opbroek AAW, Benfield JR, van Krieken JHJH, Dijkman
JH. 1997. The alveolar type II cell is a pluripotential stem cell in the
genesis of human adenocarcinomas and squamous cell carcinomas.
Histol Histopathol 12:319 –336.
Woodworth CD, Wang H, Simpson S, Alvarez-Salas LM, Notario V.
1993. Overexpression of wild-type p53 alters growth and differentiation of normal human keratinocytes but not human papillomavirus-expressing cell lines. Cell Growth Differ 4:367–376.
Yin Y, Tainsky MA, Bischoff FZ, Strong LC, Wahl GM. 1992. Wildtype p53 restores cell cycle control and inhibits gene amplification
in cells with mutant p53 alleles. Cell 70:937–948.
Yonish-Rouach E, Resnitzky K, Lotem J, Sachs L, Kimchi A, Oren M.
1991. Wild-type p53 induces apoptosis of myeloid leukemic cells
that is inhibited by interleukin 6. Nature 352:345–347.
Yonish-Rouach E, Grunwald D, Wilder S, Kimchi A, May E, Lawrence
JJ, May P, Oren M. 1993. P53-mediated cell death: relationship to
cell cycle control. Mol Cell Biol 13:1415–1423.