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JOURNAL OF PATHOLOGY, VOL.
178: 249-254 (1996)
EVIDENCE FOR DISSOCIATION OF HISTONE mRNA
EXPRESSION FROM CELLULAR PROLIFERATION IN
CUTANEOUS HUMAN PAPILLOMAVIRUS INFECTION
STEPHEN
A. HINCHLIFFE",
MARTIN D.
SMITH?,MATIIILDE
E.
JONATHAN
BOON$,c. VIVYAN HOWARD§,
DICK
L. R E E S ~
VAN V E L Z E N ~AND
*Department of Pathology, University of Newcastle upon Tyne, U.K.; ?Department of Dermatology, University of
Newcastle upon Tyne, U.K.; fLeiden Cytology and Pathology Laboratories, Leiden, The Netherlands;
$Department of Fetal and Infant Pathology, University of Liverpool, U.K.
SUMMARY
Replication of human papillomavirus (HPV) is thought to require the expression of components of the host cell DNA replication
apparatus. The expression of the mRNA encoding the DNA replication-dependent histones H2B, H3, and H4 was investigated in a series
of verrucae using an FITC-labelled oligonucleotide cocktail for in situ hybridization, to explore the possibility of HPV inducing the
transcription of host cell histones. In contrast to normal epidermis, in which the majority of histone mRNA labelling was confined to
basal cells, a bimodal distribution of histone positivity was observed in all verrucae. In addition to basal hyperproliferation-associated
labelling, numerous positive suprabasal cells were found within the stratum spinosum. By contrast, in another hyperproliferative lesion,
psoriasis, the labelling pattern was similar to that found in the normal controls, with characteristic basal hyperproliferation but no
evidence of suprabasal labelling. The presence of HPV replication in every verruca and its absence from all controls of normal and
psoriatic skin were demonstrated immnnohistochemically, using a polyclonal antibody to the viral capsid proteins. It is proposed that the
presence of histone mRNAs in differentiated suprabasal keratinocytes of verrucae is due to the virus turning on parts of the cellular
apparatus in order for it to replicate in the absence of cellular proliferation. The results also emphasize that when assessing proliferation
with in situ hybridization for histone mRNA, the possibility of HPV causing the labelling of non-proliferating cells should be considered.
KEY
WORDS-DNA
replication; histone mRNA; in situ hybridization; human papillomavirus; skin
INTRODUCTION
Human papillomaviruses (HPVs) are epitheliotropic,
double-stranded DNA viruses associated with a variety
of proliferative squamous lesions of the skin and
mucosal surfaces lining the anogenital tract, oral cavity,
and larynx. The oncogenic potential of certain HPV
types is reflected in their association with squamous cell
carcinoma of the skin' and uterine cervix.2
In contrast to other DNA viruses, the process of
replication of HPV is poorly understood, largely as a
consequence of the inability, until very recently, to
propagate the virus in cell culture and reproduce the
infectious ~ y c l e . ~
There
, ~ is evidence, however, that
vegetative viral DNA replication occurs only in
squamous epithelial cells undergoing terminal different i a t i ~ n . In
~ . basal
~
cell nuclei, the viral genome is stably
maintained as low copy number, monomeric, extrachromosomal plasmids. As the infected cell undergoes
terminal differentiation, the viral DNA undergoes
vegetative replication, producing thousands of copies
per cell. Late genes encoding the viral capsid proteins are
subsequently expressed and virions are a ~ s e m b l e d . ~ , ~
It has become apparent that of the many proteins
necessary for replication of viral DNA, HPV encodes
only the replication origin binding proteins El and
E2.x,9Therefore it has been postulated that HPV must
Addressee for correspondence: S. A. Hinchliffe, MB, PhD, Department of Pathology, University of Newcastle upon Tyne, Royal
Victoria Infirmary, Newcastle upon Tyne NEI 4LP, U.K.
CCC 0022-34 17/96/030249-06
0 1996 by John Wiley & Sons, Ltd.
have a mechanism for reactivation of the host cell DNA
replication apparatus, to facilitate its own replication in
cells undergoing terminal differentiation which do not
replicate their chromosomal DNA and thus presumably
no longer transcribe those genes essential for DNA
synthesis.l o
In this regard, several authors have demonstrated the
induction of proliferating cell nuclear antigen (PCNA),
an accessory protein of host DNA polymerase delta and
an essential part of the cellular replication machinery,
in differentiated suprabasal cells of HPV-infected skin
(verruca vulgaris)'
and cervix (condyloma acuminatum and low-grade cervical intraepithelial neoplasia),1°
which was independent of the oncogenic risk potential
of the infecting HPV genotype. These data support the
hypothesis of an interaction between HPV and host cell
replication processes and provides impetus for a study of
other cellular genes associated with DNA synthesis.
Recently, a technique has been described for the
detection of the messenger RNA (mRNA) transcripts of
several cellular histones by in situ hybridization
(ISH). l37l4 Expression of the highly conserved H2A,
H2B, H3, and H4 histone genes,I5 encoding small basic
proteins involved in the folding of eukaryotic DNA, is
tightly coupled to the replicative stage of the cell
~ y c l e . ~The
~.'~
number of mRNA transcripts encoding
these replication-dependent proteins increases 30- to
100-fold on entering S-phase, due to the combination of
an approximately two- to three-fold increase in the rate
of histone gene transcription, together with an increase
in the mRNA half-life from 8-12 to 45-60 min.1x,19
As
Received I December 1994
Accepted 7 July 1995
250
S.A. HINCHLIFFE E T A L
the cell enters G2, and throughout M, G1, and GO,
transcripts are rapidly destabilized owing to the presence
of a unique stem-loop sequence in the 3' non-coding
region of the mRNA2" and the absence of a 3' polyadenylate tail.'* Non-S-phase cells thus contain a small
but undetectable pool of transcripts. Detection of these
histone transcripts by ISH may therefore be considered a specific marker for cell cycle-related DNA
replication.*'
This technique has been proposed as an additional
assessment of cellular proliferation in routinely processed tissue section^.'^.'^.^^ Results suggest that histone
mRNA ISH provides an estimation of the proportion
of cells in S-phase which is of comparable sensitivity to
that determined by flow cytometry,13 [3H]thymidine
labelling,'4,23or BrdU i n c ~ r p o r a t i o n . ~ ~
Following alkaline disruption of HPV particles purified from human plantar warts, four low-molecularweight polypeptides closely associated with the viral
capsids were resolved and noted to be similar to the
cellular histones H2A, H2B, H3, and H4.25 As we have
previously demonstrated the excellent cellular resolution
obtained using non-isotopic ISH (NISH) for the detection of mRNA species in skin,26 we considered it of
interest to investigate the expression of DNA
replication-dependent histone mRNAs in a series of
verrucae to explore the possibility of HPV inducing the
transcription of host cell histones.
MATERIALS AND METHODS
Specimen selection
The study group comprised 12 consecutive excision
specimens of verruca vulgaris, which had been referred
for routine histopathological examination at either the
LCPL Laboratories, Leiden ( n = 2) or the Department of
Fetal and Infant Pathology, Liverpool (n= 10). All
lesions were derived from skin of the hand or foot [five
males, seven females; mean age 15.4 (4-42) years].
In addition, control biopsies were taken at the
Department of Fetal and Infant Pathology, Liverpool
and at the Department of Dermatology, Newcastle of
normal skin [n= 16; ten males, six females; mean age 16.4
( 8 4 9 ) years] and of psoriatic plaques, as an example of
a non-HPV-associated hyperproliferative disorder [n= 5;
three males, two females; mean age 48.8 (28-66) years].
None of these individuals had a history of cutaneous
HPV infection.
Informed consent was obtained in all cases prior to
biopsy.
Tissue processing
The specimens received at the LCPL Laboratories,
Leiden were fixed by immediate immersion in Kryofix
(Merck, Darmstadt, Germany), a non-crosslinking
agent,z7followed by processing to paraffin in a conventional 650 W microwave oven.28The specimens received
at the Department of Fetal and Infant Pathology,
Liverpool and at the Department of Dermatology,
Newcastle were fixed by immersion in 0.1 M phosphate
buffered, pH 7.4 per cent formaldehyde for a minimum
of 6 h and a maximum of 12 h, and subsequently
routinely processed to paraffin.
Non-isotopic in situ hybridization (NISH) for histone
mRNA
Each of the 12 verruca vulgaris specimens, 16 normal
skin biopsies, and five biopsies from areas of psoriatic
plaque formation were analysed for the presence of
histone mRNA by NISH using a newly available commercial, fluoroscein-labelled oligonucleotide cocktail
complementary to unique sequences on the mRN A
transcripts encoding the replication-dependent human
histones H2B, H3, and H4.
NISH was performed as previously described,26 with
the following modifications:
(i) Pretreatment-Following
hydration, 5 pm sections mounted on 3-aminopropyltriethoxysilane-coated
slides were incubated with proteinase K [5pglml in
100 mM Tris-HCI, pH 8.0, 50 mM ethylenediamine
tetraacetic acid (EDTA); 15 min at 37"C] followed by
immersion in 0.2 per cent glycine (in PBS, 5 rnin), and
two changes of PBS (5 min each). Slides were then
acetylated in 0.25 per cent acetic anhydride in 0.1 M
triethanolamine (pH 8.0) for 10 min followed by two
further rinses in PBS (5 min each).
(ii) Hybridization-Sections were allowed to air-dry
prior to the application of 2Opl of FITC-labelled histone
oligonucleotide probe cocktail (Novacastra Laboratories Ltd., Newcastle upon Tyne, U.K.). The probe was
supplied in hybridization buffer at a working concentration, to which freshly denatured salmon sperm DNA
was added to a final concentration of lOOpg/ml. Sections were hybridized for 16 h at 37°C in a chamber
humidified with 3 0 0 m ~NaCl, 30 mM sodium citrate
(2 x SSC)/30 per cent formamide.
(iii) Post-hybridization wushes and signal detection-Following hybridization, coverslips were removed and
slides washed in two changes of 2 x SSC/30 per cent
formamide at 37°C (15 min each). Following rinses in
2 x SSC (2 x 5 min) and PBS (5 min), sections were
incubated with 10 per cent normal rabbit serum (NRS)
in PBS for 20 rnin at room temperature. NRS was then
replaced with alkaline phosphatase-conjugated rabbitanti-FITC Fab' fragments (Dako, Denmark) diluted
1 : l O O in 1 per cent NRS. Following incubation for 60
min, slides were washed in two changes of PBS (5 min
each) and one change of substrate buffer (100 m M NaCl,
50mM MgCl,, p H 9.5; 5 rnin). FITC-labelled hybrids
were visualized by incubation in a chromogen solution
(substrate buffer containing 0.34 mg/ml nitrobluetetrazolium and 0.17 mg/ml5-bromo-4-chloro-3-indolylphosphate).
( i v ) Controls-Positive
control was human tonsil
which was known to exhibit characteristic strong signal
positivity. Negative controls for hybridization specificity consisted of adjacent sections which were 'sham'
25 1
NISH HISTONE mRNA AND HPV
hybridized with a buffer containing no probe, but
otherwise treated identically.
Detection of HP V capsid proteins by
immunohistochemistry
Each of the 12 verruca vulgaris specimens, 16 normal
skin biopsies, and five biopsies from areas of psoriatic
plaque formation were analysed for the presence of HPV
replication using a rabbit anti-bovine polyclonal antibody (DAKO B0580, dilution 1:lOOO) which reacts
specifically against papillomavirus genus (common)
capsid antigens, regardless of the host specie^."^.^^
Details of the immunohistochemical technique are
reported elsewhere.'
Estimation of HPV capsid protein and histone mRNA
labelling indices
Histological sections orthogonal to the basement
membrane/epidermal surface were selected. The characteristic architectural stratification of the epidermis
allowed the identification of individual keratinocyte
layers parallel to the basement membrane. In order to
incorporate in each case at least part of the stratum
granulosum, ten such layers, starting at the basal layer,
were separately assessed for the presence of HPV capsid
proteins and histone mRNA.
The labelling indices of a particular cell layer were
estimated by counting the number of positive cells
within a systematic series of randomly located samples
of ten cells in that layer. The total number of samples of
ten cells to be assessed in any particular layer in a given
specimen was determined using the continuous running
mean method.
In all layers of every verruca, fewer than ten samples
of ten cells were found to be sufficient to reach a mean
within 5 per cent of the final mean. Therefore for the
estimation of labelling indices in this group, ten randomly located samples of ten cells were assessed for the
number of cells positive for HPV capsid proteins and
histone mRNA signal (a total of 100 nuclei per cell
layer).
In the analysis of histone mRNA positivity in normal
and psoriatic biopsies, a stable mean within 5 per cent
of the final mean required the counting of a maximum of
30 samples of ten cells per layer. Therefore estimation of
the labelling index in these specimens was based on the
analysis of 30 such samples, a total of 300 nuclei per cell
layer. No staining for HPV capsid proteins was seen in
any of the normal or psoriatic biopsies.
Labelling index was expressed as the number of
positive cells/total number of epidermal cells assessed
x 100 per cent.
Reproducibility analysis
Intra- and inter-observer reproducibility were studied
by repeated, blind assessment of the labelling index by
two different observers.
Statistical analysis
Comparison of labelling indices was performed using
paired t-tests.
RESULTS
Histone NISH gave a high cellular resolution with
little non-specific staining; all 'no probe' controls were
clear of signal background. There was no appreciable
difference in the staining quality or the labelling index
between the two groups of verrucae processed in the
Liverpool and Leiden laboratories. Intra- and interobserver reproducibility of the labelling index estimation
were greater than 93 per cent for every sample. Photomicrographs of representative areas of HPV-infected,
psoriatic, and normal skin are shown in Fig. 1.
The results of histone NISH labelling index estimation
are shown in Fig. 2. A similar pattern of positivity was
observed in normal and psoriatic skin, with labelling
confined to the basal and immediately suprabasal cells.
The mean labelling index was significantly greater in the
psoriatic skin (mean layers 1-3,3.10 per cent versus 1.04
per cent WO.01). In marked contrast, the verrucae
displayed a bimodal pattern of histone positivity: the
mean labelling index decreased from 15.1 per cent (layer
1) to 0 per cent (layer 3) (P<O.OOl), increased to 30.5 per
cent (layer 5 ) (P<O.OOl), then decreased to 5.9 per cent
(layer 6) (P<O.OOl). Histone positivity in the lowermost
layers of the verrucae (mean layers 1-3, 7-30 per cent)
greatly exceeded that of the normal skin (mean layers
1-3, 1.04 per cent) (P<O.OOl).
Immunohistochemistry using the antibody to HPV
capsid proteins produced staining of suprabasal cell
nuclei in all verrucae, as reported in detail elsewhere."
HPV capsid proteins were first detected in epidermal
layer 3; the labelling index increased in the more superficial layers, with positivity of the vast majority of nuclei
in layers 7-10. The relationship between the labelling
indices for HPV capsid proteins and histone mRNA is
shown in Fig. 3. No staining for HPV capsid proteins
was observed in any of the normal or psoriatic control
biopsies.
DISCUSSION
This study confirms the applicability of non-isotopic
in situ hybridization for the detection of histone
mRNA transcripts in archival formalin-fixed, paraffinembedded tissue, as reported by other^.'^,'^,^^,^^
In normal skin, labelling for the replication-dependent
histones was restricted to occasional basal and immediately suprabasal cells, in agreement with our previous
The verrucae showed a bimodal labelling pattern (Fig.
2). In keeping with the epidermal hyperproliferation
characteristic of this lesion, significantly more basal cells
contained histone mRNAs than in normal, age-matched
skin. The second peak of histone labelling in the differentiated suprabasal keratinocytes (layers 4 6 ) of the
stratum spinosum was a striking finding and not
observed in the psoriatic specimens. In the latter lesion,
epidermal hyperproliferation, a feature of both psoriasis
and verrucae, was present but restricted, as reported by
others, to the lowermost cell layers.31 Similarly, on
histological examination of the verrucae, the very
252
S . A. HINCHLIFFE ET A L
Fig. 1-Photomicrographs of non-isotopic in situ hybridization for the mRNA transcripts encoding cellular histones H2B, H3, and H4 in
HPV-infected (A), psoriatic (B), and normal (C) skin. Note the bimodal pattern of the signal in HPV-infected skin (A), with positive cells
present in the stratum spinosum (layers 4 and 5) in addition to the most basal layers. The signal in sporiatic (B) and normal (C) skin is
confined to the most basal layers
++-
~
W
verruca vulgaris
- histone
A-
psoriarir
HPV
,*-k--A
/
cn
=
-c
40
al
n
a
i
20
0
1
2
3
4
5
6
7
8
B
10
Epidermal Cell Layer
-I
L
0
/
4
I
I
II
1
2
3
4
5
6
7
8
L
9
10
Epidermal Cell Layer
Fig. 2-The relationship between histone mRNA labelling index and
epidermal cell layer in HPV-infected, psoriatic, and normal skin. Data
are expressed as mean ( f SEM)
Fig. 3-The relationship between histone mRNA labelling index and
HPV capsid protein labelling index in vet-rucae. HPV data are
published elsewhere"
occasional mitotic figure was noted in the stratum
spinosum, but the vast majority of mitotic activity
resided within basal cells.
Suprabasal histone positivity in verrucae thus appears
to be associated with the presence of HPV and is not
merely a reflection of epidermal hyperproliferation. This
conclusion is supported by Penneys et al., who used
seborrhoeic keratoses as a control for non-HPV-related
epidermal hyperproliferation in their study of PCNA
expression in cutaneous HPV infection. I 2 Although they
detected PCNA in cells of the stratum spinosum in
verrucae, staining in the seborrhoeic keratoses was confined to the basal layers. In this study, it was not possible
to determine which specific HPV genotypes were present
253
NISH HISTONE mRNA AND HPV
in each verruca. However, we consider that the association between suprabasal histone positivity and HPV is
unlikely to be type-specific, since others have demonstrated different viral types, notably 1, 2, and 4, in
individual lesions.32
We propose that the presence of histone mRNA
transcripts in differentiated suprabasal keratinocytes of
verrucae is associated with HPV replication as distinct
from cellular proliferation, although the possibility of
the latter cannot be entirely excluded. HPV must have a
mechanism to reactivate this and other host cell replication genes, to facilitate the amplification and subsequent packaging of its own DNA in cells that have
exited from the cell cycle and are undergoing terminal
differentiation.
Examination of Fig. 3 shows, using data from our
previous study,' that in verrucae the appearance of
large amounts of HPV capsid antigens detectable by
immunohistochemistry is spatially tightly associated
with the suprabasal peak of replication-dependent histone mRNAs. Vegetative viral replication would thus
appear to be restricted to a specific subpopulation of
keratinocytes at a particular stage of differentiation.
Subsequently the more superficial keratinocytes, whilst
continuing to contain large amounts of virus, no longer
transcribe detectable levels of histone mRNA and have
presumably ceased to replicate viral DNA.
The concept of viral replication occurring only within
a subset of keratinocytes is supported by other in vivo
studies. In both skin (verruca vulgaris)" and cervix
(condyloma acuminatum and low-grade cervical
intraepithelial neoplasia),1° co-detection of PCNA and
HPV DNA was restricted to a relatively narrow band of
the epithelium; more superficial cells contained the virus,
but PCNA induction could not be demonstrated. Similarly, recent in vitro experiments have demonstrated
tight linkages between the expression of both low-risk
HPV-11 and high-risk HPV-16 viral E6 and E7 genes,
induction of PCNA, and amplification of viral DNA in
epithelial raft cultures of differentiated keratin~cytes~
and in organotypic cultures of dysplastic keratinocyte~.~~
In summary, we have presented evidence confirming
the applicability of in situ hybridization for replicationdependent histone transcripts to routinely fixed and
embedded tissue. HPV replication in the epidermis was
strongly associated with the de nova transcription of one
or more of the host cellular histones H2B, H3, and H4.
Viral replication would thus appear to involve not only
the reactivation of host cell repIication mechanisms such
as DNA polymerases and associated proteins (e.g.,
PCNA), but also a dependence on the host for the
proteins with which, following replication, the viral
chromosomes are complexed. Taken together, the
results of this and other recent studies lead us to
speculate that regulation of the viral life cycle is in some
way intimately related to the degree of differentiation of
the infected cell.
Finally, when assessing cellular proliferation with
histone in situ hybridization, one must be aware of the
possible labelling of non-proliferating cells by HPV and
perhaps other viral infections.
ACKNOWLEDGEMENTS
We are grateful to Dr J. Seymour for comments on a
previous draft of the manuscript. MDS is supported by
the Medical Research Council, U.K.
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