Hypoxia is associated with poor prognosis in squamous cell carcinomas affecting both
local control and distant spread (Hockel et al., 1996a, 1996b, 1999; Nordsmark et
al, 1996; Fyles et al, 2002; Kaanders et al, 2002). Local control is believed to depend
on local radiation response while distant spread is thought to depend, at least in
part, on the induction of oxygen-regulated proteins. In order to test this, pimonidazole,
an extrinsic marker for tissue hypoxia (Arteel et al, 1995; Kennedy et al, 1997; Varia
et al, 1998; Raleigh et al, 1999), with prognostic value (Kaanders et al, 2002) was
used to examine whether ORPs such as VEGF (Raleigh et al, 1998a), metallothionein
(Raleigh et al, 2000), HIF-1α (Janssen et al, 2002), Glut-1 (Airley et al, 2003) and
CAIX (Olive et al, 2001) were, in fact, associated with cellular hypoxia in human
tumours. Unexpectedly, VEGF and metallothionein (MT) were not expressed in the majority
of hypoxic cells in squamous cell carcinomas (Raleigh et al, 1998a, 2000) even though
these ORPs were induced by hypoxia in experimental systems (Shweiki et al, 1992; Raleigh
et al, 1998b; Murphy et al, 1999).
A possible explanation for this apparent anomaly was found in reports that VEGF and
MT are expressed in oxygenated basal lamina of normal stratified epithelia and not
in more differentiated, suprabasal layers farthest from the blood vessels (Quaife
et al, 1994; Sundelin et al, 1997; Viac et al, 1997). This led to the conclusion that
VEGF and MT are downregulated by differentiation in stratified epithelia (Quaife et
al, 1994; Viac et al, 1997). In analogy with their untransformed counterpart, squamous
cell carcinomas often express markers for terminal differentiation in the centre of
tumour nests farthest from blood vessels (Roland et al, 1996). Pimonidazole binding
was known to occur in these regions (Kennedy et al, 1997), and subsequent studies
demonstrated that the majority of the hypoxic cells express involucrin, a molecular
marker for epithelial cell differentiation (Raleigh et al, 2000). It was concluded,
therefore, that the lack of VEGF and MT expression in hypoxic cells was due to downregulation
by differentiation. At the same time, it appeared that involucrin might be an oxygen-regulated
protein.
Involucrin is a 96 kDa cell envelope protein that appears in free form in the early
stages of keratinocyte terminal differentiation. During the late stages of differentiation,
involucrin is crosslinked with proteins and lipids to form cornified cell envelopes
in the uppermost cells of stratified epithelia (Eckert and Welter, 1996). Five AP-1
consensus sites exist in the promoter region of the involucrin gene with two sites
accounting for 80% of promoter activity (Crish et al, 2002). One site is proximal
while the other is distal to the transcription start site. The proximal site is regulated
via a mitogen-activated protein kinase pathway that includes PKC, Ras, MEKK1, MEK3
and p38/RK (Efimova et al, 1998). Gel supershift analyses show that junB, junD and
Fra-1 are the major AP-1 transcription factors regulating involucrin expression (Eckert
and Welter, 1996). However, cotransfection of involucrin promoter constructs with
c-jun and c-fos can increase involucrin promoter activity, indicating that c-Jun also
stimulates involucrin transcription (Takahashi and Iizuka, 1993; Efimova et al, 1998).
Although involucrin had not been identified previously as an ORP, c-Jun/AP-1 is known
to be responsive to hypoxia in squamous cell carcinoma cells (Bandyopadhyay et al,
1995; Laderoute et al, 2002) and it was conceivable that involucrin expression was
oxygen regulated.
The present investigation examines whether involucrin is oxygen regulated in an in
vitro model comprising moderately differentiated SCC9 and poorly differentiated SCC4
squamous cell carcinoma cells (Rheinwald and Beckett, 1980, 1981). The model was of
interest because Rice et al (1988) had shown that involucrin increases spontaneously
in postconfluent cultures of SCC9 cells. Hypoxia is generated in unstirred, high-density
cell cultures (Boag, 1969; Whillans and Rauth, 1980; Jones, 1985; Kaluz et al, 2002)
and, although other explanations are possible, it seemed that involucrin might be
induced by hypoxia in the SCC9 cultures.
Investigations of potentially useful endogenous markers of hypoxia such as CAIX and
Glut-1 have shown that immunostaining for these proteins extends beyond the edges
of pimonidazole binding (Olive et al, 2001; Kaanders et al, 2002; Airley et al, 2003).
Many oxygen-regulated processes are half maximally induced at oxygen partial pressures
(K
m=6–20 mmHg) (Leith and Michelson, 1995; Jiang et al, 1996; Chiarotto and Hill, 1999;
Wykoff et al, 2000) that would strongly inhibit the binding of nitroimidazole hypoxia
markers such as pimonidazole, EF5 and misonidazole (K
m=0.8–2.0 mmHg) (Franko et al, 1987; Arteel et al, 1995; Gross et al, 1995; Koch et
al, 1995). This could account for the immunostaining patterns for Glut-1 and CAIX.
If involucrin were also induced in the range of 6–20 mmHg, it might be expressed in
tumour microregions that did not bind detectable levels of pimonidazole. In order
to explore this possibility, the K
m for involucrin expression has been measured in suspension cultures of SCC9 cells.
In poorly differentiated squamous cell carcinomas, involucrin immunostaining is generally
weak even in tumour regions that avidly bind pimonidazole (Azuma et al, 2003). This
would appear to be inconsistent with oxygen regulation. However, the transcription
status of oxygen-regulated genes can be coregulated by differentiation (Webster et
al, 1990; Claffey et al, 1992; Quaife et al, 1994; Levy and Kelly, 1997; Viac et al,
1997) and the effect of differentiation on involucrin expression was therefore examined
by comparing its expression in moderately differentiated SCC9 and poorly differentiated
SCC4 cells exposed to acute and chronic hypoxia.
MATERIALS AND METHODS
Chemicals
The hypoxia marker, pimonidazole hydrochloride (Hypoxyprobe™-1; Chemicon International
Inc., Temecula, CA, USA), was used as previously described (Arteel et al, 1995; Kennedy
et al, 1997; Varia et al, 1998; Raleigh et al, 1999, 2000). 4-Nitrophenyl phosphate
(alkaline phosphatase substrate), phosphate-buffered saline (PBS) pellets, foetal
bovine serum and hydrocortisone (cat # H-0396) were obtained from Sigma (St Louis,
MO, USA). Liquid 3,3′-diaminobenzidine (DAB) peroxidase substrate was obtained from
DAKO Corp (Carpinteria, CA, USA). Aqueous 2% formalin was obtained from Polysciences,
Inc. (Warrington, PA, USA). Enzyme-grade polyoxyethylene ether (Brij 35), polyoxyethylenesorbitan
monolaurate (Tween 20), tris(hydroxymethyl)aminomethane (Tris), Biomeda Crystal/Mount,
ProbeOn Plus glass slides and miscellaneous reagent-grade chemicals were obtained
from Fisher Scientific Company (Norcross, GA, USA). Aqua Haematoxylin was obtained
from Innovex Biosciences (Richmond, CA, USA). Gas tanks containing certified quantities
of oxygen and 5% CO2 balanced with nitrogen were purchased from National Welders Supply
Company, Inc. (Raleigh, NC, USA).
Immunological reagents
Supernatant from hybridoma clone 4.3.11.3 containing antipimonidazole IgG1 monoclonal
antibody at a concentration of 70 μg ml−1 (Chemicon International Inc., Temecula,
CA, USA) was used for the immunohistochemical detection of protein adducts of reductively
activated pimonidazole as described previously (Arteel et al, 1995; Kennedy et al,
1997; Varia et al, 1998). Diluted aliquots of rabbit polyclonal antipimonidazole antisera
were used for the enzyme-linked immunosorbent assay (ELISA) of pimonidazole binding
to cell lysates (Arteel et al, 1995). A biotin-conjugated F(ab′)2 fragment of a rabbit
anti-mouse IgG was obtained from Accurate Chemical Scientific Corp. (Westbury, NY,
USA) and used as the secondary reagent for the immunohistochemical detection of pimonidazole
binding. Protein blocker and peroxidase-conjugated streptavidin were obtained from
DAKO Corp. An IgG1 mouse anti-human involucrin antibody clone SY5 used for the immunohistochemical
detection of involucrin was obtained from Sigma. An ELISA kit containing rabbit anti-human
involucrin antisera and affinity-purified goat anti-rabbit IgG conjugated to alkaline
phosphatase used to detect involucrin in cell lysates were obtained from Biomedical
Technologies Inc. (Stoughton, MA, USA).
Confluent cell culture
SCC9 and SCC4 cell lines derived from a squamous cell carcinoma of the human tongue
(American Type Culture Collection, Rockville, MD, USA) were grown in Dulbecco's modified
eagle medium/F12 containing 1.0 mM.calcium ion concentration and supplemented with
10% foetal bovine serum, 0.4 μg ml−1 of hydrocortisone and 14 mM of sodium bicarbonate.
Cells were seeded at a density of 3 × 105 cells in 100-mm diameter culture dishes.
Every 3 days, the culture medium was exchanged with fresh medium containing 100 μ
M pimonidazole hydrochloride as hypoxia marker. Cell samples were harvested at 1,
4, 6, 9 and 12 days after confluence. Harvested cells were washed three times with
cold PBS and cell densities were measured by cytometry. Cells were lysed in cold buffer
containing 0.2 mM EDTA, 10 mM Tris, 0.5% Triton X-100 and 200 μl/106 cells of proteinase
inhibitors (1.0 μg ml−1 of Leupeptin, 1.0 μg ml−1 of pepstatin and 1 mM phenylmethylsulphonyl
fluoride) (Gaido and Maness, 1994). Cell lysates were stored at –80°C until they were
analysed by ELISA for pimonidazole adducts and involucrin.
Immunostaining confluent cultures for involucrin and pimonidazole adducts
SCC9 cells were added to a six-well tissue culture plate at a density of 105 cells
per well. Each well contained four 22 mm square cover glass slides to which the cells
attached. At 2 days prior to confluence, at confluence and 5 days after confluence,
cells on the glass slides were fixed with 2% of formaldehyde in PBS for 20 min. The
slides were washed and permeabilised with 0.02% of saponin in PBS containing 5% of
serum-free protein block for 30 min. Fixed cells were incubated with antipimonidazole
IgG1 monoclonal antibody 4.3.11.3 (1 : 50) and anti-human involucrin monoclonal antibody
(1 : 100) for 1 h. The cells were then incubated with biotin-conjugated rabbit anti-mouse
F(ab′)2 IgG antibody (1 : 500) for 30 min . The cells were incubated with streptavidin-conjugated
peroxidase for 20 min and colour developed by incubation with DAB for 10 min. The
cells were counterstained with haematoxylin at room temperature for 25 s and washed.
The cover slides were placed on a microscope slide, with the cells facing the surface
of the microscope slide and mounted with CrystalMount.
Exposure of cells to hypoxia in suspension culture
When SCC9 and SCC4 cells reached confluence they were trypsinised and collected. Aliquots
of 5 × 106 cells in 25 ml of culture medium containing 100 μ
M pimonidazole hydrochloride and 1.0 mM calcium ion were added to 250 ml glass vessels
fitted with PTFE inlet and outlet stopcocks and a small diameter injection port (cat.#
7401–50; Ace Glass, Inc., Vineland, NJ, USA). In order to minimise cell adhesion,
the vessels were silanised by treatment with Sigmacote (Sigma, St Louis, MO, USA)
followed by extensive washing with distilled water. Cells were kept in suspension
by attaching the gassing vessel to the deck of an orbital shaker (Model SS110504;
Integrated Separation Systems, Natick, MA, USA) in a warm room maintained at 37°C.
The system was flushed for 20 min in order to remove oxygen dissolved in nonglass
components of the system. These included two PTFE stopcocks, a small red rubber septum
port, PTFE unions connecting gas wash bottles to nylon transmission tubing (12723
Universal Connector; Ace Glass, Inc.), short lengths of flexible tygon tubing that
connected reciprocating glass tubes to stiff, low-permeability 3/16 inch internal
diameter nylon transmission tubing (A-06489-06; Cole-Palmer Instrument Co., Vernon
Hills, IL, USA) and 150 ml of distilled water in a gas wash bottle used to humidify
the gas stream. Following flushing, the system was subjected to 12 rounds of partial
vacuum followed by pressurisation with gas phases containing 10, 100, 500, 5000, 10 000,
20 000 or 25 000 ppm oxygen and 5% CO2 balanced with nitrogen. The gas exchanges –
which were carried out over a period of 5 min – facilitated the rapid equilibration
of molecular oxygen in gas and aqueous phases. Once equilibrated, cells were incubated
with shaking under a continuous flow of gas. Previous studies showed that cell viability
is not affected by this procedure (Arteel et al, 1995). Teflon and nylon have low
oxygen permeability (see Cole-Palmer Instrument Company 2003/04 catalogue, p.1910)
and once flushed were not expected to be a source of oxygen contamination. Rubber
and tygon are more permeable, but oxygen contamination from rubber septa and short
lengths of tygon tubing was also considered to be insignificant in a system equilibrated
and then continuously flushed with a flow of gas. Whillans and Rauth (1980) have shown
that continuous flushing following equilibration is adequate to control pO2 down to
at least 0.01% even when relatively long sections of tygon tubing are used.
The K
m experiment was repeated twice and the data points averaged for both pimonidazole
binding and involucrin expression. Control experiments showed that the presence of
pimonidazole did not affect involucrin expression. Cells were collected, washed three
times with cold PBS and cell densities were measured by cytometry. Cells were lysed
and stored at –80°C for subsequent ELISA analysis for involucrin and pimonidazole
adducts.
ELISA
The ELISA for pimonidazole adducts followed a previously published method for 2-nitroimidazole
hypoxia markers (Raleigh et al, 1994; Thrall et al, 1994; Arteel et al, 1995), except
that cell lysates were prepared by homogenisation without pronase K digestion. Briefly,
100 μl well−1 of serial dilutions of cell lysates and serial dilutions of pimonidazole
hydrochloride standards were incubated for 1 h at 37°C in 96-well microtitre plates
containing 100 μl well−1 of rabbit polyclonal antipimonidazole antisera diluted 6 : 10 000
in PBS-Tween (0.05% Tween 20 in PBS). The mixtures were transferred to ELISA plates
coated with a Ficoll-pimonidazole conjugate as solid-phase antigen and the plates
incubated for 1 h at 37°C. The plates were washed with PBS-Tween by means of an Ultrawash
plate washer (Dynex Technologies Inc., Chantilly, VA, USA); 100 μl well−1 of a 1 : 2000
goat anti-rabbit antibody conjugated with alkaline phosphatase was added, and the
plates were incubated for 1 h at 37°C. The plates were washed and 100 μl well−1 of
a 1 mg ml−1 solution of alkaline phosphatase substrate dissolved in 10% diethanolamine
pH 9.8 buffer was added. Colour development at 405 nm was followed for 5 min by means
of a Molecular Devices plate reader. Kinetic data were analysed by means of Vmax DeltaSoft
3 software (Biometallics, Inc., Princeton, NJ, USA). ELISA data were corrected for
the fact that pimonidazole hydrochloride, although a convenient standard, is 25 less
effective as a competitive inhibitor than protein adducts of pimonidazole (Arteel
et al, 1995). The data were normalised to cell lysate protein content as measured
by the Bio-Rad Dc protein assay (Bio-Rad, Hercules, CA, USA) using bovine serum albumin
as a standard.
The involucrin ELISA kit was used according to the directions provided by Biomedical
Technologies Inc. In the final step, a secondary goat anti-rabbit IgG-conjugated alkaline
phosphatase was used to detect the binding of the antiinvolucrin rabbit antisera to
involucrin solid phase antigen. End point colour development at 405 nm associated
with the hydrolysis of 4-nitrophenyl phosphate was recorded after 30 min. ELISA data
for involucrin were normalised for protein content in the cell lysates using bovine
serum albumin as a standard.
Clinical samples
Contiguous tumour sections immunostained for involucrin and pimonidazole binding were
available from head and neck squamous cell carcinomas from an earlier study (Raleigh
et al, 2000). The study had received local Institutional Review Board approval for
the type of experiment described here. Patients enrolled in the study had signed informed
consent forms prior to their participation in the study (Raleigh et al, 2000). The
Hypoxyprobe-1 used for the clinical studies was obtained from NPI, Incorporated (Belmont,
MA, USA).
RESULTS
Involucrin and hypoxia in confluent cultures of SCC9 and SCC4 cells
ELISA measurements revealed a steady increase in involucrin expression in SCC9 cells
beginning ca 4 days postconfluence. The increase in involucrin expression was associated
with an increase in pimonidazole binding in the cultures. The maximum involucrin protein
expression occurred ca 9 days postconfluence (Figure 1
Figure 1
Involucrin expression (solid circles) and pimonidazole binding (open circles) in moderately
differentiated SCC9 cells growing in confluent culture in the presence of 1.0 mM calcium
ion. The increase in involucrin expression more or less parallels that for pimonidazole
binding. The involucrin data are similar to those reported by others (Rice et al,
1988).
). Pimonidazole binding increased in confluent cultures of SCC4 cells but, unlike
SCC9 cells, a corresponding increase in involucrin did not occur (Figure 2
Figure 2
Involucrin expression (solid circles) and pimonidazole binding (open circles) in poorly
differentiated SCC4 cells growing in confluent culture in the presence of 1.0 mM calcium
ion concentration. Little or no involucrin is induced in the cultures, even though
pimonidazole binding indicates the presence of hypoxia in the cultures.
).
Immunostaining of postconfluent SCC9 cell cultures showed punctate patterns for both
involucrin expression and pimonidazole binding. That is, the whole culture was not
hypoxic but rather subsets of cells within the cultures formed pimonidazole adducts
(data not shown). Owing to piling up in the cultures, it has not been possible to
determine whether pimonidazole-positive cells are also involucrin positive by microscopic
examination of dual stained slides (data not shown).
K
m for involucrin expression in suspension cultures of SCC9 cells
Involucrin induction was half maximal at a gas phase oxygen concentration of approximately
20 000 ppm for SCC9 cells harvested at the point of confluence and exposed to graded
concentrations of oxygen for 2 h (Figure 3
Figure 3
K
m curves for involucrin (solid circles) and pimonidazole binding (open circles) in
SCC9 cells exposed for 2 h to different pO2 in the presence of 1.0 mM calcium ion
concentration. The data represent averages of two independent experiments. Range of
data for the two experiments is shown where it exceeded the dimension of the data
symbol. Note the 40-fold difference in K
m between involucrin induction and pimonidazole binding and the steep pO2 dependence
for involucrin induction.
). A gas phase concentration of 20 000 ppm is equivalent to a partial pressure of
15 mmHg, a gas phase concentration of 2% or a dissolved oxygen concentration of 21 μ
M at 37°C. Cells harvested 9 days postconfluence, when involucrin expression was at
a maximum, showed no additional induction of involucrin during acute exposure to hypoxia
in suspension culture. As was the case in confluent cultures, involucrin was not induced
in SCC4 cells during hypoxic exposure in suspension culture.
The K
m for involucrin induction was ca 40 times higher than that for pimonidazole binding
in SCC9 cells (Figure 3). Furthermore, the oxygen dependence for involucrin expression
in SCC9 cells was much steeper than that for pimonidazole binding, increasing from
minimum to maximum over less than one order of magnitude change in pO2 compared to
pimonidazole binding that rose from minimum to maximum over two orders of magnitude
as expected for a competition between pimonidazole and oxygen for reducing equivalents
(Arteel et al, 1998).
Immunostaining patterns for involucrin and hypoxia in head and neck squamous cell
carcinomas
Figure 4
Figure 4
Immunostaining patterns for involucrin expression (left panels) and pimonidazole binding
(right panels) in contiguous sections from squamous cell carcinomas (SCC) of the head
and neck. (A and B) Immunostaining for involucrin and pimonidazole adducts, respectively,
in sections from a Grade 1 floor of the mouth SCC. (C and D) Immunostaining for involucrin
and pimonidazole adducts in sections from a Grade 2 larynx SCC. Immunostaining for
involucrin (A) extends well beyond that for pimonidazole binding (B) and is expressed
in the absence of pimonidazole binding in some microregions. (C and D) Immunostaining
for involucrin and pimonidazole adducts is closely matched with the extent of involucrin
immunostaining covering an area ca 1.5 that for pimonidazole. (E and F) Immunostaining
for involucrin and pimonidazole adducts in sections from a Grade 3 larynx SCC. Little
or no involucrin expression is observed in the presence of extensive pimonidazole
binding. Original magnification: × 12.5.
shows representative examples of immunostaining for involucrin expression and pimonidazole
binding in contiguous sections taken from squamous cell carcinomas of the head neck.
Figure 4A and B are derived from a well-differentiated (Grade 1) squamous cell carcinoma
of the floor of the mouth. In this case, immunostaining for involucrin extends well
beyond the edges of immunostaining for pimonidazole adducts and, in some regions,
involucrin is expressed in the absence of pimonidazole binding. Figure 4C and D are
derived from a moderately differentiated (Grade 2) tumour of the larynx. In this case,
the extent of immunostaining for involucrin conforms more closely to that for pimonidazole
adducts, with the extent of immunostaining differing by a factor of only ca 1.5. Figure
4E and F are derived from a poorly differentiated (Grade 3) squamous cell carcinoma
of the larynx. In this case, little or no involucrin is expressed even in the presence
of substantial amounts of pimonidazole binding.
DISCUSSION
Involucrin expression increases with increasing hypoxia in confluent cultures of SCC9
cells consistent with the idea that involucrin is an oxygen-regulated protein. The
induction of involucrin during hypoxic exposure of SCC9 cells in suspension culture
confirms that involucrin can be induced by hypoxia in moderately differentiated squamous
cell carcinoma cells. Induction occurs over a period of 2 h and is, therefore, relatively
rapid. In contrast to SCC9 cells, hypoxia induces little or no involucrin in poorly
differentiated SCC4 cells in spite of the fact that the involucrin gene is reported
to be functional with ample quantities of involucrin mRNA present in these cells (Gibson
et al, 1996). Interestingly, Gibson et al observed a distinction between well-differentiated
keratinocytes and poorly differentiated SCC4 cells with respect to calcium ion-induced
involucrin expression. In particular, high calcium concentration induced involucrin
mRNA and protein in keratinocytes but not in poorly differentiated SCC4 cells (Gibson
et al, 1996). This similarity between calcium and hypoxia regulation might be important
for understanding how hypoxia induces involucrin. For example, Salnikow et al (2002)
have described an HIF-1α independent pathway for the hypoxia induction of AP-1 regulated
genes in which hypoxia-induced intracellular calcium release and subsequent interaction
at AP-1 promoter sites are key events. Calcium ion concentration is known to increase
in the outer layers of stratified epithelia (Denda et al, 2000) and it is conceivable
that hypoxia interacts with calcium ions to stimulate the production of involucrin
and other AP-1-dependent proteins. Experiments are underway to test whether the effect
hypoxia on involucrin induction is direct or one mediated by intracellular calcium
ions. It should be noted that the process of differentiation itself is initiated in
the well-oxygenated basal cells of stratified epithelia (Watt, 1983) and is unlikely,
therefore, to be initiated by hypoxia.
Involucrin induction has a time course similar to that for pimonidazole binding but
is distinguished by a very steep dependence on pO2. A similarly steep dependence has
been reported for VEGF mRNA induction in a number of cell lines (Chiarotto and Hill,
1999). This steepness of response is reminiscent of synergistic interactions and it
is tempting to speculate that these might involve interactions between hypoxia and
calcium ions reported for the case of VEGF (Salnikow et al, 2002). Involucrin induction
is further distinguished from pimonidazole binding in that the K
m (15 mmHg) is similar to that for VEGF (Leith and Michelson, 1995; Chiarotto and
Hill, 1999) and, therefore, ca 40 times higher than that for pimonidazole binding
(0.4 mmHg). The rapid rate of induction, steep pO2 dependence and high K
m could be important in understanding immunostaining patterns for involucrin in squamous
cell carcinomas.
In well-differentiated tumours, immunostaining for involucrin is more extensive than
that for pimonidazole binding and in some areas, involucrin is expressed in the absence
of pimonidazole binding (Figure 4A and B; Azuma et al, 2003). Superficially, a 40-fold
difference in K
m for involucrin induction and pimonidazole binding might account for this. However,
it does not explain why immunostaining for involucrin more closely matches pimonidazole
binding in moderately differentiated tumours (Figure 4C and D) where the extent of
immunostaining differs by a factor of only ca 1.5. A similar small factor of ca 2
has been reported for the difference between the extent of CAIX expression and pimonidazole
binding in squamous cell carcinomas (Olive et al, 2001). One explanation is that different
levels of acute hypoxia exist in the two tumours (Pigott et al, 1996). That is, the
more extensive immunostaining for involucrin is due to rapid induction during acute
changes in hypoxia that pimonidazole binding cannot match. However, both pimonidazole
binding and involucrin are easily detected within 2 h of hypoxic exposure in vitro
making this explanation less likely. A second possibility is that hypoxia developed
during the time between pimonidazole washout (plasma t
1/2=ca 5 h) and tumour biopsy, but this would require a major change in oxygen distribution
in the tumour depicted in Figure 4A and B, which seems unlikely.
A third possible explanation for immunostaining patterns in Figure 4 is that oxygen
gradients are steeper in moderately differentiated tumours than in well-differentiated
tumours. Steep oxygen gradients would foreshorten the distance over which divergent
K
ms are traversed, possibly reducing the distance to the two or three cell diameters
observed in moderately differentiated tumours (Figure 4C and D) and in normal tissues
such as the liver and kidney (Arteel et al, 1995; Zhong et al, 1998). Conversely,
shallow gradients would increase the distance over which divergent K
ms are traversed allowing for involucrin induction in advance of pimonidazole binding
as appears to be the case in Figure 4A and B. Variations in the slope of oxygen gradients
have been proposed to account for the lack of nitroimidazole binding around areas
of necrosis in a subset of glioma xenografts (Parliament et al, 1997; Franko et al,
1998; Turcotte et al, 2002), but it remains to be seen whether this occurs among subsets
of squamous cell carcinomas.
Poorly differentiated tumours express very little involucrin even in the areas of
extensive pimonidazole binding. This is observed for squamous cell carcinomas of the
head and neck (Figure 4E and F) and uterine cervix (Azuma et al, 2003). While this
does not appear to be consistent with oxygen regulation, it matches in vitro data
where involucrin is induced by hypoxia in moderately differentiated SCC9 cells but
not in poorly differentiated SCC4 cells. While there is no basis for believing that
the mechanism that prevents involucrin induction in SCC4 cells is exactly the same
as that which prevents involucrin expression in the hypoxic regions of poorly differentiated
squamous cell carcinomas, the model system does show that dedifferentiation can suppress
involucrin induction by hypoxia.
Hypoxia inhibits differentiation in some cell lines (Sahai et al, 1997) and possibly
in breast carcinomas (Helczynska et al, 2003), but the association between hypoxia
and involucrin expression (Figures 1, 3 and 4) indicates that this might not be true
for squamous cell carcinomas. It is important to emphasise, however, that involucrin
is an early marker for terminal differentiation so that tumour hypoxia, while not
totally inhibiting differentiation, might arrest it at some point short of end stage
differentiation. Cell lines derived from a poorly differentiated squamous cell carcinoma,
for example, can express involucrin without losing proliferative capability (Auersperg
et al, 1989). This is also true of SCC9 cells where confluent cells are easily subcultured
in spite of possessing substantial levels of involucrin. Clearly, the presence of
involucrin need not be a sign of end stage differentiation and further work will be
required to define the extent to which hypoxic cells in squamous cell carcinomas are
differentiated. This could be important because it is known that differentiation increases
radiosensitivity in human carcinoma cells under both hypoxic and aerobic conditions
(Hallows et al, 1988; Hoffmann et al, 1999). Radiosensitisation might be due to inhibited
DNA repair arising from the limited access of DNA repair machinery in differentiated
cells (Wheeler and Wierowski, 1983). To the extent that the in vitro results apply
clinically, hypoxic cells that are more differentiated might be less radioresistant
than otherwise thought.
A recurring theme in the study of endogenous hypoxia markers – whether it be HIF-1α,
CAIX, Glut-1 or involucrin – is heterogeneity of expression (Olive et al, 2001; Wiesener
et al, 2001; Haugland et al, 2002; Janssen et al, 2002; Kaanders et al, 2002; Airley
et al, 2003). The basis for heterogeneity in the case of involucrin appears to be
related to cell differentiation. In the case of HIF-1α, functional inactivation of
the von Hippel Lindau factor might be the most important factor (Wiesener et al, 2001;
Haugland et al, 2002; Janssen et al, 2002; Turner et al, 2002). Endogenous ORPs appear
to be useful as hypoxia markers in normal tissues (e.g. Lee et al, 2001), but without
a good understanding of the factors that control ORP expression, heterogeneity of
expression of proteins such as involucrin will limit their scope as markers of human
tumour hypoxia.