| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Clinical Trials |
Departments of Radiation Oncology [J. A. R., S-C. C., D. P. C-A., C. A. B., M. A. V.] and Pathology [D. B. N.], University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
MTs are a family of Mr 6000 proteins comprised of MT-I, MT-II, MT-III, and MT-IV classes with multiple isoforms within each class (12, 13, 14, 15) . MT-I and MT-II are ubiquitously expressed and are stress inducible (13 , 15) . MT-I isoform inducibility is reported to depend on the embryonic germ layer from which a tumor is derived. For example, tumors originating from intermediate mesoderm, such as cervical carcinomas, exhibit MT-II and MT-If genes in an inducible form, whereas the MT-Ie gene is refractory to induction. MT-If inducibility is associated with poor prognosis, whereas MT-Ie inducibility is associated with better prognostic features in human tumors (13) . MTs regulate intracellular concentrations of zinc and other metal ions. MT overexpression, therefore, can influence transcription, replication, and protein synthesis and might explain why MT overexpression is associated with high-grade tumors (16, 17, 18, 19, 20) , including carcinomas of the head and neck (21) .
Murphy et al. (22 , 23) have shown that MT-I and MT-IIa can be induced by hypoxia and that induction in vitro is regulated by metal transcription factor-1. The possibility that the effects of tumor hypoxia are mediated by MT is supported by the report that MT is induced by hypoxia in experimental tumors (24) and that MT is overexpressed in human tumors possessing low median pO2 (25) . However, it was not known from the clinical study whether MT was induced by hypoxia. This is an important point because MT can be regulated by both hypoxia and proliferation (26, 27, 28) . Under these circumstances, a technique for discriminating hypoxic from proliferating cells was needed. The immunohistological hypoxia marker approach (29) , based on pimonidazole hydrochloride combined with proliferating cell nuclear antigen as a histological marker for proliferation (11 , 30, 31, 32, 33) , was considered ideal for an investigation of the relationship between hypoxia and MT expression in human tumors.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Pimonidazole Hydrochloride.
Pimonidazole hydrochloride (Hypoxyprobe-1) was obtained from NPI, Inc.
(Belmont, MA) in the form of 1.0-g samples of lyophilized white powder
contained in sealed vials. Ten ml of sterile 0.9% saline were added to
the solid, and a volume of the solution equivalent to a calculated
patient dose of 0.5 g/m2 was withdrawn and added
to 100 ml of sterile 0.9% saline. The diluted solution was infused
i.v. over 20 min.
Squamous Cell Carcinomas.
University of North Carolina at Chapel Hill Institutional Review Board
approval was obtained for the clinical use of pimonidazole
hydrochloride, and protocol eligibility requirements were identical to
those published previously (33)
.
Tumor Biopsies.
Sixteen to 24 h after pimonidazole hydrochloride infusion,
multiple biopsies were obtained from geographically distinct areas of
the tumors. Biopsies were obtained from all quadrants of the tumor
whenever possible, but regions of obvious necrosis were avoided. On
average, four biopsies (range, one to nine) were obtained, and tissue
sections were analyzed for each of the markers of interest. Biopsy
selection and analyses were based on previous sampling error studies
(34
, 35) . One section per biopsy was considered adequate
for the purposes of the present study.
Fresh biopsy samples were placed in cold, 10% neutral buffered formalin, held at 4°C for 12–24 h, processed into paraffin blocks, and the blocks were sectioned. Four-µm-thick sections were placed on ProbeOn Plus slides. One tissue section per biopsy was stained with hematoxylin to confirm the presence of tumor. Separate sections were immunostained for pimonidazole adducts, MT-I/II, PCNA, and involucrin.
Immunohistochemistry.
For illustrative purposes, immunostaining for the different factors was
performed on four contiguous sections from a single biopsy (Fig. 1
). However, for the qualitative and
quantitative analyses in Tables 1
and 2
, two sets of two contiguous sections
from each biopsy were prepared in which one section in each set was
immunostained for pimonidazole adducts, and the second section in each
set was immunostained for either MT-I/II or involucrin. There were 84
sets of contiguous sections for MT and hypoxia analysis and 83
evaluable sets for involucrin and hypoxia analysis.
|
|
|
Tumor tissue sections were immunostained for MT-I/II in a manner similar to that used for pimonidazole adducts. A 40°C Pronase antigen retrieval step was included, but the incubation time with Pronase was shortened to 20 min for both cervix and head and neck tumors. According to the manufacturer’s literature, the commercially available IgG1 mouse antihorse MT antibody (clone E9; used in 1:50 dilution) was raised against horse, self-polymerized MT-I and MT-II. The antibody binds to the 5-amino acid moiety (AcMet-Asp-Pro-Asn-Cyst-) at the end of the NH2 terminus of the B domain of equine and human MT-I and MT-II (37) . The anti-MT antibody does not distinguish between MT-I and MT-II, and therefore, the designation MT-I/II is used to characterize immunostaining for MT in the present report. The NH2 terminus of MT-IV (AcMet-Asp-Pro-Gly-Glu-Cyst-; Ref. 15 ) differs significantly from that of MT-I/II, and it has been reported that MT-I/II antibodies do not cross-react with MT-IV (13) . MT-III expression is restricted to brain tissue (12) and was not a concern for the present study.
Immunostaining for PCNA was performed in a manner similar to that for
pimonidazole adducts, except that Pronase digestion was omitted and
goat antimouse immunoglobulin conjugated to a peroxidase-labeled
dextran polymer (DAKO EnVision+ reagent) was used as the secondary
reagent. PCNA is a Mr 36,000
nonhistone nuclear protein, the expression of which is associated with
late G1, S, and early G2
phases of the cell cycle. It is an auxiliary protein to DNA polymerase
and plays a critical role in the initiation of cell proliferation.
PCNA expression has been correlated with bromodeoxyuridine uptake in
human tumors, and the characteristics of PCNA immunostaining can be
used to identify S-phase cells in tissue sections (33)
.
According to the manufacturer’s literature, the IgG2a monoclonal
anti-PCNA antibody (used in a 1:100 dilution) was raised against rat
PCNA made in the protein A expression vector pR1T2T.
Immunostaining for involucrin was carried out in a manner similar to that for pimonidazole adducts, except that Pronase antigen retrieval was omitted from the immunostaining procedure. Involucrin is a cytoplasmic Mr 92,000 protein that is cross-linked to other proteins by the action of transglutaminase during terminal differentiation of keratinocytes (38) . According to the manufacturer’s literature, the IgG1 mouse antihuman involucrin antibody clone SY5 (used in a 1:100 dilution) was raised against purified human involucrin.
Negative controls, in which primary antibodies were omitted from the protocols, showed no nonspecific binding attributable to secondary antibody reagents. Positive controls containing pimonidazole adducts, MT-I/II, PCNA, and involucrin showed staining patterns and intensities that were constant from staining session to staining session. Samples of normal human tongue served as a positive control for involucrin.
Qualitative Analysis.
Microregional comparisons of hypoxia with MT and involucrin expression
were carried out on a section-by-section basis without respect to
whether the squamous cell carcinoma occurred in the cervix or head and
neck on the assumption that MT induction is independent of tumor site.
Tissue sections from the biopsies, one per biopsy, were qualitatively
assessed for hypoxia, MT-I/II, and involucrin expression. Each section
was viewed at x100, and individual microscopic fields (190 x 190
µm) were assessed with respect to: (a) no overlap;
(b) overlap; or (c) a mix of overlap and no
overlap with immunostaining for pimonidazole adducts.
Quantitative Analysis.
Quantitative image analysis was carried out for pimonidazole adducts
and MT-I/II. Color detection threshold and default width settings were
chosen for the 3,3'-diaminobenzidine chromogen on the basis of an
immunostained region at x200 in tissue sections. The settings for the
chromogen staining were optimized for intensity, saturation, and hue so
that all cells that were identified as labeled above background by
visual inspection were also scored by the image analysis software.
Cells immunostained above a threshold intensity were scored as labeled,
with no distinction being made between light and heavy immunostaining.
This approach provides the number of tumor cells that are labeled,
which is of paramount importance, and obviates concerns about
variations in marker binding intensity arising from individual
differences in pharmacokinetics, tumor cell redox properties, and time
to biopsy. It was assumed that all cells labeled with pimonidazole
adducts irrespective of immunostaining intensity were at
pO2 < 10 mm Hg and were, therefore, of interest
with respect to increased radiation resistance (35
, 36)
.
Multiple fields 1–60(1–60, depending on section size) from each section
from each biopsy were captured at x200 by means of an Axioskop 50
microscope (x10) and Fluar objective (x20; Carl Zeiss, Inc.,
Thornwood, NY) linked through a high resolution, three-chip video
camera (Carl Zeiss, Inc.; model ZVS-3C750E) to a high resolution Sony
Trinitron RGB color monitor (model PVM 1343 MD) and a 486/33
microprocessor workstation running Image-1 software (Universal Imaging
Corp., West Chester, NY). Mean percentage of area immunostained in each
field was calculated as a percentage of the total field area minus
areas of acellularity, necrosis, and stroma.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
are representative of immunostaining
patterns for pimonidazole adducts (Fig. 1A
), MT-I/II (Fig. 1B
), PCNA (Fig. 1C
), and involucrin (Fig. 1D
). A comparison of A and B shows
little or no overlap between hypoxia and MT-I/II. This is most clearly
seen in the upper right of the photomicrographs, where staining for
pimonidazole adducts in A is in a zone separate from that
for MT-I/II staining in B. The arrows in the
photomicrographs identify the periphery of the tumor nest and help in
comparing immunostaining patterns. A comparison of B and
C in Fig. 1
reveals that MT-I/II is expressed primarily in
cells in the periphery of the tumor nests that also stain for S-phase
PCNA.
A comparison of A and C in Fig. 1
shows that
there is little or no overlap between cells that are stained for
S-phase PCNA and those stained for pimonidazole adducts. This is
consistent with previous studies in which immunostaining for hypoxia
markers and PCNA have been compared (32
, 33)
. A comparison
of A and D in Fig. 1
shows that involucrin
staining overlaps with that for pimonidazole adducts to a remarkable
extent.
Of the total number of 84 sets of tissue sections used for the analysis
of hypoxia and MT expression, 64 were found to contain hypoxia. Of
these, 43 of 64 (67%) possessed little or no overlap between hypoxia
and MT. That is, these sections had patterns like those shown in
A and B of Fig. 1
. Of the remaining sections, 7
of 64 (11%) possessed overlap, and 14 of 64 (22%) possessed a mixture
of overlap and no overlap in the same section (Table 1)
. When the
analyses were performed on a tumor-by-tumor basis, a complete absence
of overlap between MT and hypoxia occurred in 5 of 7 head and neck
tumors and in 7 of 13 cervix tumors.
Of the total number of 83 sets of tissue sections that were available
for the analysis of hypoxia and involucrin, 72 contained measurable
hypoxia. Of these, 59 of 72 (82%) showed overlap between
immunostaining for pimonidazole adducts and involucrin. Of the
remaining sections, 10 of 72 (14%) showed a mixture of fields with
overlap and no overlap and 3 of 72 (4%) showed no overlap at all
(Table 1)
. The lack of overlap in individual fields was attributable
entirely to microregions of hypoxia that did not express involucrin.
However, when all 83 sections were analyzed including those that had no
detectable hypoxia, it was found that 11 of 83 (13%) sections showed
involucrin expression in the absence of hypoxia, as defined by the
absence of pimonidazole binding. When examined on a tumor-by-tumor
basis, 4 of 7 head and neck and 7 of 13 cervix tumors tissue sections
possessed involucrin and hypoxia overlap as the only patterns of
immunostaining. In the remaining tumors, sections showed mixed
patterns of overlap and no overlap.
Quantitative Analysis.
Patient stage and grade are summarized in Table 2
. The data are
organized in ascending order of tumor hypoxia. No trends between
hypoxia and stage or grade were observed. Hypoxia was measurable in all
except two tumors [tumors nos. 1 and 2 in Table 1
(<0.1% area
hypoxic)]. The range of the percentage of area hypoxia was wide for
both head and neck and cervix tumors, 0.9% to 17% and <0.1 to 14%,
respectively. Areas immunostained for MT-I/II were measurable in all
but one tumor (tumor 10 in Table 1
) and ranged widely from 0.0 to
36.6%. No correlation between overall hypoxia and MT was discernible
(Fig. 2)
.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Transcriptional control for both VEGF and MT is complex and, in the case of hypoxic induction, subject to different hypoxia-sensitive transcription factors. The fact that neither VEGF nor MT is expressed in the majority of hypoxic cells in squamous cell carcinomas indicates that a generalized mechanism for the suppression of oxygen-regulated protein expression might be operating. One possibility is that hypoxic regions defined by pimonidazole binding are ischemic. Shweiki et al. (43) have shown, for example, that combined severe glucose and oxygen depletion suppresses the expression of VEGF in rat C6 glioma cells in monolayer culture. Although this mechanism cannot be ruled out, Arteel et al. (44) have shown that the binding of hypoxia markers requires energy, and it seems unlikely that pimonidazole-labeled cells are severely nutrient depleted. A second possibility is that pimonidazole binds to cysteine residues in oxygen-regulated proteins, thereby inhibiting their detection by antibodies (45) . The 20-amino acid epitope at the NH2 terminus of VEGF is cysteine free, but the 5-amino acid epitope at the NH2 terminus of MT-I/II contains a cysteine moiety. However, this does not appear to be a problem in practice because MT-I/II protein was easily detectable in pimonidazole-labeled cells of rodent tumors (24) with the antibody used in the present study. Consideration of other possible global mechanisms led us to reports that keratinocyte differentiation suppresses the expression of VEGF (46) and MT (15) and that squamous carcinomas express markers for terminal differentiation (47) .
During normal stratified epithelial maturation, keratinocytes move from
the proliferating, basal layer into suprabasal layers, where synthesis
of specific cytoskeletal proteins occurs. As keratinocyte
differentiation proceeds, cells are pushed through the stratum spinosum
and stratum granulosum toward the outermost stratum corneum, where they
become flattened cytoskeleton-filled scales or squames. Morphological
changes are accompanied by the expression of molecular markers for
terminal differentiation such as involucrin, transglutaminase, and a
variety of cytokeratins (48)
. Details of the molecular
biology of these changes in squamous cells is under active
investigation (49)
. Interestingly, molecular markers for
terminal differentiation are also expressed in squamous cell carcinomas
(Fig. 1)
, and it is generally accepted that the markers are associated
with differentiation processes in the tumors (50, 51, 52, 53, 54, 55, 56, 57, 58)
.
The remarkable colocalization of involucrin and pimonidazole adducts
raises questions of whether involucrin is an oxygen-regulated protein
and whether oxygen gradients regulate differentiation. Although there
are no published data on this point, there is evidence that
differentiation events are initiated in the well-oxygenated basal cell
layer of normal stratified epithelia and that cell migration to
suprabasal layers is a consequence, and not a cause, of differentiation
(48)
. If this is correct, then the expression of
involucrin in hypoxic cells of squamous cell carcinomas is
coincidental. Whether oxygen gradients play a role in intensifying the
involucrin signal seen in Fig. 1D
is not known. It is
generally accepted that patients with hypoxic and poorly differentiated
tumors, as defined pathologically, have poorer prognoses. The tight
association between hypoxia and involucrin expression would seem
paradoxical in this regard. However, pathological assessment of
differentiation makes use of morphological clues in tissue sections,
and it is possible that tumor cell differentiation can, in many cases,
proceed to a point that falls short of producing changes that are
visible under the microscope (50)
.
As reported by others and shown in Fig. 1
, MT-I/II immunostaining
occurs in proliferating microregions in the periphery of tumor nests
(59)
, as might be expected of a protein that is associated
with cell cycle progression (26)
. On the other hand,
MT-I/II immunostaining is largely absent from hypoxic regions in the
center of tumor nests where involucrin is strongly expressed (Fig. 1
;
Refs. 47
, 54,
and 55
). Quaife et
al. (15)
have found that MT gene
expression switches from highly inducible MT-I to noninducible MT-IV
when cells transition from the basal layer to more differentiated
layers in normal stratified epithelia, and it is tempting to speculate
that the absence of MT-I/II protein expression in hypoxic cells in
squamous cell carcinomas is attributable to the suppression of the
inducible forms of MT by cell differentiation in hypoxic zones. It has
been shown in studies of explanted cell lines from human cervical
carcinomas that cells expressing involucrin are not necessarily
undergoing a process of normal differentiation (50)
, and
it remains to be seen whether changes in the expression of MT isoforms
are correlated with the microregional expression of involucrin in
squamous cell carcinomas. In situ hybridization studies of
the microregional distribution of human MT-I, MT-IIa, and MT-IV mRNA
expression are under way to test this.
Relevance to Cancer Therapy.
The expectation that MT expression in the hypoxic cells of human tumors
might add to the radioresistance (60, 61, 62, 63)
or
chemoresistance (64, 65, 66)
of these cells is not supported
by the present results. However, MT in proliferating compartments of
tumors could account for the inverse relationship between overall MT
expression and chemotherapy response (21
, 67
, 68)
by both
protecting cells near blood vessels and limiting drug diffusion beyond
perivascular regions (69)
. With respect to radiation
therapy, it is interesting to note that differentiation sensitizes
human tumor cells in vitro (70
, 71)
. Both oxic
and hypoxic cells are sensitized, and their relative radiosensitivities
are, therefore, unchanged. However, if hypoxic cells in squamous cell
carcinomas were more differentiated than oxic cells, then hypoxic cells
might be selectively radiosensitized and their therapeutic importance
diminished. The importance of hypoxic cells in squamous cell carcinomas
would be further diminished if they were differentiated to the extent
that they were incapable of reentering the cell cycle. Given these
observations and the conventional wisdom that differentiated tumor
cells belong to the cell loss compartment in tumors (72)
,
further investigations of the extent to which hypoxic cells in squamous
cell carcinomas are differentiated appear to be warranted.
Summary.
The present results combined with earlier data for VEGF indicate that
the expression of oxygen-regulated genes in human squamous cell
carcinomas at the time of clinical presentation is not predicted by
in vitro studies with tumor cell lines. The reason for this
is not clearly understood, but a possible scenario is that
differentiation of the hypoxic cells exerts collateral control on gene
expression whereby oxygen-regulated protein expression is suppressed.
The lack of MT expression in hypoxic tumor cells indicates that this
radio- and chemoprotective protein will not contribute directly to the
therapy resistance of hypoxic cells in squamous cell carcinomas.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Financial support for this study was supplied by
Department of Health and Human Services Grants CA68826, CA74069, and
RR00046 and the State of North Carolina. 
2 To whom requests for reprints should be
addressed, at Department of Radiation Oncology, University of North
Carolina School of Medicine, CB# 7512, Chapel Hill, NC 27599. Phone:
(919) 966-7710; Fax: (919) 966-7681; E-mail: raleigh{at}radonc.unc.edu
3 The abbreviations used are: VEGF, vascular
endothelial growth factor; HIF, hypoxia-inducible factor; MT,
metallothionein; PCNA, proliferating cell nuclear antigen.
Received 10/12/99; revised 12/23/99; accepted 12/30/99.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
in common human cancers and their metastases. Cancer Res., 59: 5830-5835, 1999.
, and P-glycoprotein in curatively resected gastric cancer. Oncology, 54: 391-399, 1997.[Medline]
B: potential role in metallothionein-mediated mitogenic response. Cancer Res., 58: 2335-2338, 1998.This article has been cited by other articles:
|
|
 |
|
  L. E. Via, P. L. Lin, S. M. Ray, J. Carrillo, S. S. Allen, S. Y. Eum, K. Taylor, E. Klein, U. Manjunatha, J. Gonzales, et al. Tuberculous Granulomas Are Hypoxic in Guinea Pigs, Rabbits, and Nonhuman Primates Infect. Immun., June 1, 2008; 76(6): 2333 - 2340. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Patiar and A. L Harris Role of hypoxia-inducible factor-1{alpha} as a cancer therapy target Endocr. Relat. Cancer, December 1, 2006; 13(Supplement_1): S61 - S75. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. R. Laderoute, K. Amin, J. M. Calaoagan, M. Knapp, T. Le, J. Orduna, M. Foretz, and B. Viollet 5'-AMP-Activated Protein Kinase (AMPK) Is Induced by Low-Oxygen and Glucose Deprivation Conditions Found in Solid-Tumor Microenvironments. Mol. Cell. Biol., July 1, 2006; 26(14): 5336 - 5347. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Franco, S. Man, L. Chen, U. Emmenegger, Y. Shaked, A. M. Cheung, A. S. Brown, D. J. Hicklin, F. S. Foster, and R. S. Kerbel Targeted Anti-Vascular Endothelial Growth Factor Receptor-2 Therapy Leads to Short-term and Long-term Impairment of Vascular Function and Increase in Tumor Hypoxia. Cancer Res., April 1, 2006; 66(7): 3639 - 3648. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Solomon, D. Binns, P. Roselt, L. I. Weibe, G. A. McArthur, C. Cullinane, and R. J. Hicks Modulation of intratumoral hypoxia by the epidermal growth factor receptor inhibitor gefitinib detected using small animal PET imaging Mol. Cancer Ther., September 1, 2005; 4(9): 1417 - 1422. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. O. Arcasoy, K. Amin, S.-C. Chou, Z. A. Haroon, M. Varia, and J. A. Raleigh Erythropoietin and Erythropoietin Receptor Expression in Head and Neck Cancer: Relationship to Tumor Hypoxia Clin. Cancer Res., January 1, 2005; 11(1): 20 - 27. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. E. Knies-Bamforth, S. B. Fox, R. Poulsom, G. I. Evan, and A. L. Harris c-Myc Interacts with Hypoxia to Induce Angiogenesis In vivo by a Vascular Endothelial Growth Factor-Dependent Mechanism Cancer Res., September 15, 2004; 64(18): 6563 - 6570. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Azuma, S.-C. Chou, R. A. Lininger, B. J. Murphy, M. A. Varia, and J. A. Raleigh Hypoxia and Differentiation in Squamous Cell Carcinomas of the Uterine Cervix: Pimonidazole and Involucrin Clin. Cancer Res., October 15, 2003; 9(13): 4944 - 4952. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. L. Olive, C. Aquino-Parsons, S. H. MacPhail, S.-Y. Liao, J. A. Raleigh, M. I. Lerman, and E. J. Stanbridge Carbonic Anhydrase 9 as an Endogenous Marker for Hypoxic Cells in Cervical Cancer Cancer Res., December 1, 2001; 61(24): 8924 - 8929. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||
