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Regular Articles |
From the Departments of Oncology*
and
Pathology,
Hadassah-Hebrew University
Hospital, Jerusalem, and InSight Ltd.,
Rabin
Science Park, Rehovot, Israel
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among these enzymes is an endo-ß-glucuronidase (heparanase) that cleaves heparan sulfate (HS) at specific intrachain sites.4-6 HSPGs are ubiquitous macromolecules associated with the cell surface and ECMs of a wide range of cells and tissues.7,8 The basic HSPG structure consists of a protein core to which several linear HS chains are covalently linked.7,8 The ability of HS to interact with ECM macromolecules such as collagen, laminin, and fibronectin and with different attachment sites on the cell membrane suggests a key role for this proteoglycan in the self-assembly and insolubility of ECM components, as well as in cell adhesion and locomotion.9-11 HSPGs are prominent components of blood vessels.12 In capillaries they are found mainly in the subendothelial basement membrane where they support the vascular endothelium and stabilize the structure of the capillary wall. Cleavage of HS, therefore plays an important role in extravasation of blood-borne cells.4,5
Previous studies performed by us and by other groups have demonstrated a correlation between the expression of heparanase and the metastatic potential of various tumor cell lines.4,5 Moreover, heparanase activity was detected in the urine of patients with aggressive metastatic cancer but not in the urine of healthy donors.13 Treatment of experimental animals with heparanase inhibitors (eg, nonanticoagulant species of low molecular weight heparin, polysulfated saccharides) markedly reduced the incidence of experimental metastases.4,5,14,15
Apart from its involvement in the egress of cells from the vasculature,
heparanase may play an accessory role in angiogenesis and tissue repair
by releasing HS-bound growth factors16-18
and promoting
endothelial cell migration and basement membrane degradation. Recently,
partial sequencing of heparanase purified from human placenta,
platelets, and hepatoma cells, followed by screening of expressed
sequence tag (EST) databases led to the cloning of a cDNA and
gene encoding the heparanase protein.19-22
Only one
sequence was identified, consistent with the notion that this is the
dominant endoglucuronidase in mammalian tissues.19-22
The
genomic locus which encodes heparanase spans 
40 kb. It is composed
of 12 exons separated by 11 introns and is localized on human
chromosome 4q21.3.19,20,23
Expression of the cloned cDNA
in insect and mammalian cells yielded 65-kd and 50-kd recombinant
proteins. The 50-kd enzyme represents a N-terminal-processed enzyme
which is at least 200-fold more active than the full-length 65-kd form.
Processing was readily demonstrated during incubation of the
full-length recombinant enzyme with intact or lysed tumor
cells.19
Nonmetastatic murine T-lymphoma cells transfected
with the heparanase gene acquired a highly metastatic phenotype
in vivo, reflected by a high rate of mortality because of
massive liver infiltration of subcutaneously inoculated lymphoma
cells.19
Preferential expression of the heparanase gene
was found in metastatic cell lines and specimens of human breast,
colon, and liver carcinomas.19
In the present study we applied antisense digoxigenin-labeled heparanase RNA probes and monoclonal anti-human heparanase antibodies to investigate the heparanase gene and protein distribution at various stages of human colon carcinoma progression. Expression of the heparanase gene and protein was detected at early stages of neoplasia, already at the stage of adenoma, whereas expression in the adjacent normal-looking colon was confined to nerves, ganglion cells, and smooth muscle around blood vessels. Gradually increasing expression of heparanase was evident as the cells progressed from severe dysplasia through well-differentiated to poorly differentiated colon carcinoma. Deeply invading colon carcinoma cells and adjacent desmoplastic stromal fibroblasts showed the highest levels of the heparanase mRNA and protein. Both the heparanase gene and protein were also highly expressed in colon carcinoma metastases to lung, liver, and lymph nodes, as well as in the accompanying stromal fibroblasts.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Routinely processed formalin-fixed and paraffin-embedded specimens
from 17 patients with colonic neoplasia operated on during 1996 to 1999
were retrieved from the files of the Departments of Pathology at the
Hadassah University Hospital (Jerusalem) and the Tel-Aviv Sourasky
Medical Center. The specimens included 16 cases of adenocarcinoma, five
of which also had metastases to regional lymph nodes and three with
distant metastases (two to lungs and one to liver). Two of the patients
also had neoplastic polyps with severe dysplasia in their resected
specimen. Another patient who underwent colectomy for tubulovillous
adenoma was included. The specimens were evaluated according to
standard criteria as detailed in Table 1
.
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Riboprobe Preparation and in Situ Hybridization
A 618-bp fragment of human heparanase was subcloned into the vector pBluescript KSII multiple cloning site (+/-) (Stratagene, La Jolla, CA). The vector was linearized and used as a template for in vitro transcription of antisense or sense (control) riboprobes using T7 and T3 RNA polymerase (Promega, Madison, WI), respectively. Riboprobes were labeled with digoxigenin RNA-labeling mixture (Boehringer-Mannheim, Mannheim, Germany). In situ hybridization was performed as described previously19,24 with minor modifications. Briefly, 5-µm sections were mounted on SuperFrost Plus slides (Manzel-Glaser, Braumschweig, Germany), dewaxed, and rehydrated. Sections were first denatured with 0.2 N HCl for 10 minutes and then digested with proteinase K (20 µg/ml) at 37°C for 30 minutes. Digestion was stopped with two changes of H2O. Slides were prehybridized and hybridized, as described.24 Probe concentration was 2 µg/ml. Washes after hybridization, incubation with anti-digoxigenin antibodies, and colorimetric detection were performed as described before.24 Some of the slides were counterstained with Mayer’s hematoxylin.
Immunohistochemistry
Immunohistochemistry was performed as described before with minor modifications.19 Briefly, 5-µm sections were deparaffinized and rehydrated. Tissue was then denatured for 3 minutes in a microwave oven in citrate buffer (0.01 mol/L, pH 6.0). Blocking steps included successive incubations in 0.2% glycine, 3% H2O2 in methanol, and 5% goat serum. The first two steps were followed by two washes in phosphate-buffered saline (PBS). Sections were incubated with a monoclonal mAb (92.4) anti-human heparanase antibody diluted 1:3 in PBS, or with Dulbecco’s modified Eagle’s medium supplemented with 10% horse serum as control, diluted as above, followed by incubation with horseradish peroxidase-conjugated goat-anti-mouse IgG + IgM antibody (Jackson, Bar-Harbor, ME). mAb 92.4 is directed against the N-terminus region of the 45-kd enzyme. The preparation and specificity of this mAb were previously described and demonstrated.19 Color was developed using either Sigma Fast 3,3–diaminobenzidine tablet sets (Sigma Chemical Co., St. Louis, MO) or Zymed aminoethyl carbozole (AEC) substrate kit (Zymed, South San-Francisco, CA) for 10 minutes followed by counterstain with Mayer’s hematoxylin.
Heparanase Activity
Tissue specimens from three patients were freshly taken and homogenized in buffer containing 20 mmol/L phosphate/citrate, pH 6.0, 150 mmol/L NaCl, 1 mmol/L MgCl2, 0.1 mmol/L ZnCl2, and 0.5% Nonidet P-40. The tissue specimens were derived from the colon carcinoma and from normal-looking colon tissue away from the tumor region. The supernatant fractions were applied onto 0.3 ml of carboxymethyl (CM)-Sepharose (Pharmacia Fine Chemicals AB, Uppsala, Sweden) and the bound material was eluted with 10 mmol/L phosphate-citrate buffer, pH 6.5, supplemented with 1 mmol/L dithiothreitol, 1 mmol/L Ca++, and 0.7 mol/L NaCl. Aliquots of the eluates containing 15 µg of protein were then tested for heparanase activity using as a substrate metabolically sulfate-labeled ECM, prepared as described.5,15,19,25 Low-molecular weight sulfate-labeled HS degradation fragments released into the incubation medium were analyzed by gel filtration on a Sepharose CL-6B column.5,15,19,25 We have previously demonstrated that these fragments elute from the column at 0.5 < Kav < 0.8 (peak II, fractions 15 to 35). Nearly intact HSPG elutes just after the void volume of the column (peak I, Kav < 0.2).26 Each experiment was done at least twice and the variation of elution positions (Kav values) did not exceed ±15%.
Western Blot Analysis
For immunoblot analysis aliquots of the partially-purified material eluted from CM-Sepharose were loaded and separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins transferred to Immobilon-P membrane (Millipore, Bedford, MA), followed by successive incubations with block solution, anti-heparanase monoclonal antibodies in 1% bovine serum albumin, 10 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, and 0.05% Tween-20, and horseradish peroxidase-conjugated anti-mouse antibodies (Jackson Laboratories, Bar-Harbor, ME), as described.19 Immunoreactive bands were detected by the enhanced chemiluminescence (ECL) reagent using luminol and p-cumaric acid (Sigma, St. Louis, MO). The light emitted by the chemical reaction was detected by exposure to Hyperfilm ECL (Amersham Pharmacia Biotech, Uppsala, Sweden) for 30 to 120 seconds.19
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Monoclonal anti-heparanase antibodies were applied to detect
heparanase protein in specimens from 17 patients with colonic
neoplasia. In 15 cases the samples included apparently uninvolved
normal-looking colonic tissue, located either in close proximity or
away from the tumor, in which no heparanase stain was detected even
though in the nearby region, stained tumor cells were seen (Figure 1A)
. In all of the immunoreactive cells
staining was characterized by being both cytoplasmic and membrane
associated with many cells showing preferential cell surface
localization of the enzyme (Figure 1A
, inset), suggesting that
heparanase may be membrane associated and/or secreted. Rarely, a weak
positive staining was found on the free luminal surface of
normal-looking epithelium adjacent to the cancer (not shown). In the
majority of cases however, the surface epithelia were negative, as were
the fibroblasts in the lamina propria (Figure 1A)
. Occasional
endothelial cells in blood vessels stained weakly positive (not shown).
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, ganglion cells, and nerves (not shown), regardless of whether
tumoral or nontumoral regions were examined.
Three tubulovillous adenoma specimens were available for investigation.
Two adenomas from patients, who had also colonic carcinomas, displayed
moderate to focal severe dysplasia with superficial invasive carcinoma.
The third one showed moderate dysplasia. In all three cases very little
or no heparanase was detected in mildly dysplastic cells (Figure 1, C and D)
, whereas moderately and severely dysplastic cells expressed high
levels of the heparanase protein (Figure 1, C and D)
. The most intense
staining was found in areas of carcinomatous transformation (Figure 1D)
. Stain was negative in fibroblasts and endothelial cells, as well
as in adjacent normal-appearing mucosa (not shown). Occasional
inflammatory cells including macrophages, plasma cells, and neutrophils
were stained (Figure 1C)
.
Heparanase staining yielded a similar pattern in all cases of colon
adenocarcinoma. The more differentiated regions showed a weakly to
moderately positive heparanase expression in a proportion of the
malignant epithelial cells, whereas in poorly differentiated areas
stronger and more abundant staining was seen (Figure 1E)
. No
differences were detected between the central regions of the tumor and
areas of transition between malignant and normal-looking tissue.
However, stronger and more diffuse staining was found in deeply
invading tumor areas and was particularly prominent in desmoplastic
regions (Figure 1F)
. In these areas heparanase staining was noted also
in the proliferating fibroblasts in the stroma surrounding the invasive
cancer cells (Figure 1F)
. The stain intensity gradually increased in
both tumor cells and fibroblasts with increasing depth of invasion,
being most prominent at the leading edge of invasion and in tumor cells
in the serosal fat (not shown). Occasional stain was noted in
endothelial cells of small blood vessels in the tumor (Figure 1B)
.
In all metastases, whether to regional lymph nodes (Figure 1G)
, liver
(not shown), or lung (Figure 1H)
, moderate to marked heparanase
expression was found in the majority of metastasizing malignant
epithelial cells. Endothelial cells and fibroblasts in the metastases
were weakly positive.
Expression Pattern of the Heparanase mRNA
To investigate the correlation between the localization of the
heparanase protein and mRNA, we performed in situ
hybridization for heparanase on representative samples from four
patients with metastatic colon carcinoma, two of whom also had
synchronous tubulovillous adenomata. As shown in Figure 2
, heparanase mRNA pattern of expression
was similar to that of the heparanase protein (Figure 1)
.
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. Labeling in these areas
was confined to ganglion cells and occasional endothelial cells of
small blood vessels (not shown).
In the tubulovillous adenomas, intense heparanase-mRNA labeling was
found in dysplastic cells (Figure 2A)
. Mildly dysplastic cells were
faintly labeled (Figure 2A)
whereas the more superficially situated,
severely dysplastic cells, were heavily labeled (Figure 2A)
, similar to
the immunostaining pattern of the heparanase protein (Figure 1C)
.
Granular diffuse cytoplasmic labeling was found in malignant epithelial
cells, ranging from well-differentiated (Figure 2B)
to poorly
differentiated (Figure 2C)
grade, and from superficially (Figure 2D)
to
deeply invasive carcinoma (Figure 2E)
. Labeling of stromal cells was
only focal in the deeply invasive and less differentiated tumor areas
(Figure 2, C and 2)
. In these areas, there was labeling in occasional
endothelial cells lining small blood vessels.
As with the heparanase protein, colon carcinoma cells metastasizing
into a regional lymph node, as well as their surrounding stromal cells,
expressed the heparanase mRNA (Figure 2F)
.
Expression of Heparanase Activity and Protein
Evaluation of heparanase activity and protein levels was performed
to support the proposed involvement of the enzyme in colon
tumorigenesis. For this purpose, freshly taken specimens derived from
colon carcinoma and from normal-looking colon tissue located away from
the tumor region were homogenized and partially purified on
CM-Sepharose. Supernatant fractions were subjected to 1) sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and Western blot analysis,
and 2) evaluation of heparanase activity. As shown in Figure 3
(inset), a prominent 50-kd protein in
the tumor tissue reacted with the anti-human heparanase antibodies
(lanes 2 and 4), as compared to a very weak (lane 1) or no (lane 3)
immunoreactivity in samples derived from normal-looking colon tissue
specimens. The prominent immunoreactive protein preferentially detected
in extracts from the colon tumor further demonstrates the strict
specificity of the anti-heparanase antibodies applied in the
immunostaining studies.
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.
Degradation fragments eluted in peak II were shown to be degradation
products of HS, as they were fivefold to sixfold smaller than intact HS
side chains (Kav 0.33) released from the ECM
by treatment with either alkaline borohydride or papain; and were
resistant to further digestion with papain or chondroitinase ABC
and susceptible to deamination by nitrous acid.26
Thus,
the preferential expression of the heparanase transcripts and protein
seen by in situ hybridization and immunostaining of the
colon carcinoma is also reflected by an increased heparanase activity
found in fresh tissue samples derived from the tumor versus
the normal colon tissue. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, showing preferential expression of the
heparanase gene and protein in correlation with tumor progression.
Thus, overexpression of heparanase both in the tumor cells and
surrounding stromal fibroblasts can be regarded a characteristic
feature of colon cancer. These results are supported by the fact that
both the heparanase protein, detected by Western blot analysis, and
enzymatic activity, measured by release of HS degradation fragments
from intact ECM, were found in much higher levels in the colon tumor
tissue than in the adjacent normal-appearing tissue. The low heparanase
activity expressed by the normal tissue is most probably because of the
presence of blood inflammatory cells and platelets in the tissue
homogenate. Platelets and activated cells of the immune system
were previously shown to express a high heparanase
activity.27
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Altogether our results, using descriptive and functional approaches, support the proposed involvement of heparanase in tumor cell invasion and metastasis.4,5,14,19
The nonradioactive in situ hybridization allowed accurate identification of the cells which actually synthesize the heparanase mRNA. The co-expression of heparanase mRNA and protein implies that heparanase is synthesized and stored in the same cell, whether neoplastic epithelial cells or stromal fibroblasts. Some of the protein was detected on the cell surface, which suggests that it is membrane-associated or secreted. In case of secretion to the ECM, the protein cannot be detected because it is lost during tissue processing.
The most significant prognostic feature in colon tumors is the degree of bowel wall penetration by the tumor and the presence or absence of distant metastases,28,29 expressed by the Dukes grading system. This prognosis is also affected by the number of involved lymph nodes. Meticulous lymph node dissection, however, is often overlooked. It is believed that many patients with invasive tumor whose lymph nodes were not infiltrated and had no evidence of distant metastases but died of metastatic disease, have often had occult micrometastases that were likely missed at the time of diagnosis.30 It seems that heparanase immunostaining in the cancer cells of Dukes A and B2 samples was weaker than in Dukes C and D. In addition, there was no expression of heparanase in the stromal fibroblasts of Dukes A and one of the Dukes B2 tumors. The two other Dukes B2 specimens did express heparanase in the fibroblasts, whereas the desmoplastic stromal fibroblasts in Dukes C and D were heavily labeled. Heparanase stain may thus serve as an additional prognostic marker, and detection of strong heparanase staining may possibly allow patients selection for systemic therapies at an earlier stage. However, to verify this assumption, more cases should be investigated and followed-up for the occurrence of metastases.
Interestingly, a relatively similar correlation between the tumor stage and the expression of type IV collagenase was reported.31 The enzyme was expressed in significantly lower amounts by Dukes A and B carcinomas than by Dukes C carcinomas and was expressed even in lower levels by adenomas. In addition, type IV collagenase expression level was retained or slightly augmented in metastases compared with the matched primary tumor.31
The degree of tumor differentiation is next in prognostic importance to staging. Tumor grade correlates with the likelihood of venous and local spread, as well as with lymphatic and bowel wall penetration.32 The incidence of nodal spread increases as the tumor becomes undifferentiated and invades deeper into the bowel wall.33 Black and Waugh34 reported that 30% of low-grade adenocarcinomas metastasize to regional lymph nodes compared with 81% metastasis of high-grade lesions. The number of involved lymph nodes also increases with tumor grade. We found that heparanase expression was inversely correlated to tumor grade, being the strongest in poorly differentiated tumor areas. This suggests that heparanase may reflect the differentiation status and metastatic nature of the cells.
High-grade dysplasia represents the extreme end of the spectrum of abnormal histological changes, short of invasive carcinoma in the adenoma-carcinoma continuum. The presence of severe dysplasia strongly correlates with a contiguous invasive carcinoma.35 Although adenomas probably constitute the precursor lesions for most carcinomas, a vast gap exists in the prevalence rates of adenoma and carcinoma, indicating that 90 to 95% of adenomas will never become malignant during a person’s lifetime.36 In tubulovillous adenoma, heparanase expression gradually increased as transition of the tissue from normal through dysplastic to carcinomatous appearance took place. Heparanase expression thus correlates with the histological changes that take place during this transition. Moreover, it appears that expression of heparanase increased even as the tubulovillous adenoma progressed from mild through moderate to severe dysplasia. In addition, heparanase was not detected in normal-looking colonic epithelial cells, whereas the adjacent transformed cells were clearly positive and deeply invading tumors showed the highest level of heparanase expression. Heparanase expression pattern might thus predict the biological behavior of polyps, especially in polyps with carcinomatous transformation and invasion of the stalk. Again, the in situ hybridization and immunolocalization of the heparanase gene and protein showed the same pattern.
Overexpression of heparanase both in cancer cells and stromal fibroblasts was noted in advanced stages of tumor progression. The stromal component of colonic carcinoma varies from little fibrous stroma to frankly scirrhous tumor.37 Overexpression of heparanase by cancer cells may be because of a mutation in the heparanase gene itself or in genes regulating its expression. By expressing heparanase, the tumor cells may facilitate their ability to invade the surrounding tissue and metastasize, as previously demonstrated.19 On the other hand, fibroblasts in the tumor area are thought to be normal in the sense that they have neither mutated oncogenes nor mutated tumor-suppressor genes.38 Still, it is known that stromal fibroblasts in the vicinity of the tumor actively proliferate. Activated fibroblasts produce collagen and other ECM proteins as well as growth-promoting factors that induce the desmoplastic reaction.39,40 Overexpression of heparanase by both the tumor and connective tissue cells may contribute to this process by means of releasing heparin-binding growth factors that are stored in the ECM as a complex with HSPG.17,18,41 Expression of genes for ECM components and their degradative enzymes has been shown to be coordinated with and necessary for tissue remodeling.42 Matrix-degrading enzymes play a role in the turnover of ECM proteins of connective tissues in general43 and the stroma of pancreatic,44 gastrointestinal, and other tumors.38 Heparanase expression by stromal fibroblasts may thus be induced by the tissue remodeling that takes place during tumor progression and may reflect a physiological response to alterations in the local tissue caused by the tumor growth.
Our immunohistochemical and in situ hybridization studies point to the cancer cells as the major site of synthesis of heparanase, particularly at early stages of tumorigenesis. At later stages, the enzyme was also expressed by surrounding stromal elements such as fibroblasts. Immunohistochemistry and in situ hybridization done by other groups revealed abundant expression of urokinase in cancer cells of colon adenocarcinomas with occasional staining in stromal fibroblasts.45,46 Immunolocalization of MMP-9 and to a lesser extent MMP-2 in breast carcinoma specimens revealed staining of tumor cells compared with a weak staining of the tumor stroma.47 MMP-3, on the other hand, is specifically expressed in stromal cells of breast carcinomas.48
To our knowledge this is the first systematic study describing the progressive expression pattern of heparanase in human colon cancer. Altogether, we have shown preferential expression of the heparanase mRNA and protein in colon tumors as compared to the normal-looking colon epithelium. A correlation between the degree of tumor invasiveness, differentiation level of the cells and expression of the heparanase protein and mRNA by the carcinoma and stromal cells was noted. Expression increased as the tumor cells were more invasive and less differentiated. These results suggest a role for heparanase in colon tumor progression, most probably through its effect on the tumor microenvironment, resulting in an enhanced tumor cell invasiveness and vascularization.
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Acknowledgements |
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Footnotes |
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Supported by grants from the Israel Science Foundation founded by the Israel Academy of Sciences and Humanities; Association for International Cancer Research, United Kingdom; the Israel Cancer Research Fund; and the Israel Cancer Association.
Accepted for publication July 1, 2000.
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O. Goldshmidt, E. Zcharia, R. Abramovitch, S. Metzger, H. Aingorn, Y. Friedmann, V. Schirrmacher, E. Mitrani, and I. Vlodavsky Cell surface expression and secretion of heparanase markedly promote tumor angiogenesis and metastasis PNAS, July 23, 2002; 99(15): 10031 - 10036. [Abstract] [Full Text] [PDF]
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 R. Haimov-Kochman, Y. Friedmann, D. Prus, D. S. Goldman-Wohl, C. Greenfield, E. Y. Anteby, A. Aviv, I. Vlodavsky, and S. Yagel Localization of heparanase in normal and pathological human placenta Mol. Hum. Reprod., June 1, 2002; 8(6): 566 - 573. [Abstract] [Full Text] [PDF]
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 L. Nadav, A. Eldor, O. Yacoby-Zeevi, E. Zamir, I. Pecker, N. Ilan, B. Geiger, I. Vlodavsky, and B.-Z. Katz Activation, processing and trafficking of extracellular heparanase by primary human fibroblasts J. Cell Sci., May 15, 2002; 115(10): 2179 - 2187. [Abstract] [Full Text] [PDF]
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A. Care, F. Felicetti, E. Meccia, L. Bottero, M. Parenza, A. Stoppacciaro, C. Peschle, and M. P. Colombo HOXB7: A Key Factor for Tumor-associated Angiogenic Switch Cancer Res., September 1, 2001; 61(17): 6532 - 6539. [Abstract] [Full Text] [PDF]
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A. Koliopanos, H. Friess, J. Kleeff, X. Shi, Q. Liao, I. Pecker, I. Vlodavsky, A. Zimmermann, and M. W. Buchler Heparanase Expression in Primary and Metastatic Pancreatic Cancer Cancer Res., June 1, 2001; 61(12): 4655 - 4659. [Abstract] [Full Text] [PDF]
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O. Goldshmidt, E. Zcharia, H. Aingorn, Z. Guatta-Rangini, R. Atzmon, I. Michal, I. Pecker, E. Mitrani, and I. Vlodavsky Expression Pattern and Secretion of Human and Chicken Heparanase Are Determined by Their Signal Peptide Sequence J. Biol. Chem., July 27, 2001; 276(31): 29178 - 29187. [Abstract] [Full Text] [PDF]
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