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BRIEF REPORT
From the Department of Oncology and Neurosciences,
University G. D'Annunzio Medical School, Chieti, Italy; and the
Department of Medical, Oncological and Radiological Sciences,
University of Modena and Reggio Emilia Medical School, Modena, Italy.
A cell-adhesive protein of the human serum, 90K binds galactin-3,
Serum tumor products have become routine tools in
clinical oncologic practice, mostly as prognostic markers or for
predicting response to treatment. In lymphoma, clinical status,
computed tomography scans, and bone marrow biopsies are commonly used
to assess the response to treatment, and reliable serum markers for this purpose have not been available so far. The commonly used lymphoma
markers, serum lactate dehydrogenase and thymidine kinase, are useful
as prognostic markers when measured at the time of diagnosis. However,
these markers cannot be reliably used to predict response to treatment
because their serum levels are influenced by several factors, including
cytotoxic drugs, hematopoietic growth factors, and concurrent
disease.1
A large oligomeric protein composed of approximately 90-kd
subunits was originally identified as a tumor-secreted antigen in the
culture fluid of human breast cancer cells.2 This protein was named 90K. The sequence of 90K was elucidated after cDNA cloning and shown to contain a signal peptide and a number of cysteines and
N-glycosylation sites.3 90K was identified independently as a ligand of the lactose-specific S-type lectin, galactin-3 (formerly
known as Mac-2), and was named Mac-2 BP.4 The functions of
90K are not yet well defined but may include enhancement of killer cell
activity and cytokine production,3,5
endotoxin-Lipopolysaccharide-dependent binding to CD14,6
and tumor growth suppression.7 Other possible functions of
90K have been described, most notable of which are related to its role
in cell-cell and cell-extracellular matrix interactions. For example,
promotion of homotypic cell adhesion through cross-linking of
surface-bound galectin-3 has been documented in human melanoma
cells.8 Moreover, binding studies in vitro demonstrated
Patients
Patients with early-stage indolent lymphoma (follicular lymphoma,
extranodal marginal zone B-cell lymphoma) were treated with megavoltage
radiotherapy alone. These patients usually received single-agent
chlorambucil or cyclophosphamide if they had symptoms. Patients with
early-stage aggressive lymphoma (large B-cell lymphoma, peripheral
T-cell lymphoma) received a brief course of chemotherapy before
radiotherapy. Patients with aggressive advanced-stage disease and those
with mantle-cell lymphoma were treated with an anthracycline-containing combination chemotherapy, usually ProMACE-CytaBOM or MACOP-B. The
initial staging workup included a complete physical examination, routine blood chemistry analyses, chest and abdominal computed tomography imaging, bilateral posterior iliac crest bone marrow needle
biopsy, and laparoscopy with spleen and liver biopsy when clinically
indicated. The disease was considered bulky if the patient had a
measurable mass of at least 10 cm at its largest diameter. A complete
response was defined as the complete disappearance of all symptoms and
signs of initially documented disease for a period more than 4 weeks and the absence of new lesions as documented by negative findings
on pathology restaging procedures. Partial response was a reduction of
at least 50% in the sum of the products of the perpendicular diameters
of all measurable tumor masses for a minimum of 4 weeks without the
appearance of new lesions. Patients not included in these categories
and those who died early were considered nonresponders.
A serum sample taken at the time of diagnosis was available for each
patient. Serum samples were also obtained from 50 (30 women, 20 men)
blood donors with a median age of 42 years (range, 21 to 65 years).
Measurement of 90K
Human recombinant 90K Human recombinant 90K (hr90K) was obtained as previously described and affinity purified on an SP-2 Sepharose matrix.16Cell culture The human Jurkat T-lymphoma cell line17 was routinely cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mmol/L glutamine, and 100 µg/mL streptomycin at 37°C in an atmosphere of 5% CO2.Chemosensitivity assay First, 96-well plates were coated with 100 µL of 20 µg/mL hr90K overnight at 4°C and were blocked with 1 mg/mL bovine serum albumin (BSA; 2 hours at room temperature). Then cells (0.5-1.0 × 105) in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum and antibiotics were seeded into the wells. After 1 hour of incubation at 37°C, the cytotoxic agents doxorubicin (Sigma, St Louis, MO) and cyclophosphamide (Asta Pharmaceuticals, Frankfurt, Germany) were added to each well. Where indicated, function-blocking 1 antibody 4B4 (20 µg/mL; Coulter,
Hialeah, Florida) was added at the time of cell seeding. After 48 hours, the cells were collected and the percentage of apoptotic cells
was assessed.
Assessment of apoptosis To assess apoptosis, 2 µL of a propidium iodide (100 µg/mL)/acridine orange (100 µg/mL) (1:1 vol/vol) mixture was added to 200 µL Jurkat T cells, and the percentage of cells undergoing apoptosis was determined by fluorescence microscopy, as described.18 Apoptosis was also assessed by staining cytospin preparations using May-Grünwald-Giemsa stain.Statistics Differences between patient groups were tested with the Mann-Whitney U test or the Kruskal-Wallis test.
Circulating levels of 90K in healthy control subjects varied between 1.4 µg/mL and 16.1 µg/mL (median, 4.7 µg/mL). There was no significant difference in 90K serum concentration levels between males and females, nor was there a correlation between serum 90K levels and age or blood group. Cutoff level of serum 90K was determined to be 13.5 µg/mL, which was the mean concentration ± 2 SD. Serum 90K levels in patients with NHL (median, 14.8 µg/mL; range, 2.1-64.4 µg/mL) were significantly higher than in healthy blood donors (P < .001), and they did not statistically differ from 90K levels in patients with Hodgkin disease (P = .2). In Table 1 the percentage of patients
with serum 90K levels above the cutoff level (13.5 µg/mL) is listed
for the various groups, divided by patient characteristics. Serum level
of 90K was not associated with median age at diagnosis, gender, median serum lactate dehydrogenase level, histologic classification, Ann Arbor
clinical stage, or bulky disease (at least 10 cm in diameter). Patients
with lymphoma who had bone marrow involvement tended to have higher
serum 90K concentrations (P = .06), and this association
was significant (P = .04) among patients with NHL.
Of 129 patients evaluable for response to treatment, 97 showed
objective response, indicating an overall response rate of 75% (68 complete responses, 29 partial responses). Pretreatment 90K levels
above the normal cutoff were detected in 17 (53%) of 32 nonresponders
compared with 20 (21%) of 97 responders (P = .002; Table
2). Moreover, 90K levels were
significantly higher in nonresponders than in responders
(P = .011).
Some forms of chemotherapy, such as those used for the treatment
of lymphoma, exert their cytotoxic effects mainly by inducing apoptosis.19,20 Regulation of apoptosis in tumor cells
remains poorly understood. However, an increased level of adhesion to extracellular matrix may help tumor cells to evade the proapoptotic effects of anticancer drugs. A link between integrin-mediated cell
adhesion to matricellular proteins and protection from
chemotherapy-induced apoptosis has been reported.21,22
Because 90K is able to mediate the adhesion of tumor cells at
comparable strength with laminin,9 we evaluated whether
adhesion of lymphoma cells to 90K resulted in resistance to
chemotherapy-induced apoptosis. The addition of doxorubicin and
cyclophosphamide induced a concentration-dependent increase in Jurkat
T-lymphoma cell apoptosis, as judged by acridine orange-propidium
iodide staining and morphology. Adhesion of lymphoma cells to hr90K
substantially protected the cells against the apoptosis induced by
these chemotherapeutic agents, reducing the percentage of apoptotic
cells (Figure 1A-B). For example, in
response to 10 µmol/L doxorubicin, hr90K reduced apoptosis from 70%
to 25% (similar results were obtained with cyclophosphamide).
Nonspecific adhesion of lymphoma cells to BSA did not protect cells
from doxorubicin-induced apoptosis. Furthermore, the addition of the
In conclusion, we show here that patients with lymphoma who have
high serum 90K levels fail to respond to treatment more often than
patients with low 90K levels. Our data also provide strong preliminary
evidence that adhesion to 90K is important in lymphoma cell resistance
to chemotherapy. This occurs as a result of
We thank Mrs A. Sirotti for excellent technical assistance.
Submitted November 19, 1999; accepted July 3, 2000.
Supported in part by grants from the Associazione Italiana per la Ricerca sul Cancro 1999 and MURST Cofin ex-40% 1998.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Stefano Iacobelli, Department of Oncology and Neurosciences, University G. D'Annunzio Medical School, Via dei Vestini, 5, 66100 Chieti, Italy; e-mail: iacobell{at}unich.it.
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© 2000 by The American Society of Hematology.
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  G. Sardana, J. Marshall, and E. P. Diamandis Discovery of Candidate Tumor Markers for Prostate Cancer via Proteomic Analysis of Cell Culture-Conditioned Medium Clin. Chem., March 1, 2007; 53(3): 429 - 437. [Abstract] [Full Text] [PDF]
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 A. Grassadonia, N. Tinari, B. Fiorentino, K. Suzuki, M. Nakazato, M. De Tursi, C. Giuliani, G. Napolitano, D. S. Singer, S. Iacobelli, et al. The 90K Protein Increases Major Histocompatibility Complex Class I Expression and Is Regulated by Hormones, {gamma}-Interferon, and Double-Strand Polynucleotides Endocrinology, October 1, 2004; 145(10): 4728 - 4736. [Abstract] [Full Text] [PDF]
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 M. J. Terol, M. Tormo, J. A. Martinez-Climent, I. Marugan, I. Benet, A. Ferrandez, A. Teruel, R. Ferrer, and J. Garcia-Conde Soluble intercellular adhesion molecule-1 (s-ICAM-1/s-CD54) in diffuse large B-cell lymphoma: association with clinical characteristics and outcome Ann. Onc., March 1, 2003; 14(3): 467 - 474. [Abstract] [Full Text] [PDF]
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| Copyright © 2000 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
) or in the
presence (
) of precoated hr90K. Then increasing concentrations of
doxorubicin (A) or cyclophosphamide (B) were added. Apoptosis was
determined by propidium iodide-macridine orange or Giemsa staining.
Each point represents the mean ± SEM of 3 independent
experiments. (C) The effect of doxorubicin (10 µmol/L) on Jurkat
T-cell apoptosis in the absence of coating protein (
) and the BSA or
hr90K in the presence or absence of the function-blocking antibody 4B4.
Each bar represents the mean ± SEM.