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Technical Advance |
From the Nuffield Department of Pathology and Bacteriology,*
University of Oxford, Oxford, England; Department of Hospital
Laboratories,
University of North Carolina
Hospitals, Chapel Hill, North Carolina; the Biomarkers and Prevention
Research Branch,
National Cancer Institute,
Rockville, Maryland; the Department of Hematologic and Lymphatic
Pathology,§
Armed Forces Institute of
Pathology, Washington, DC; the Department of Thoracic
Medicine,¶
The Prince Charles Hospital,
Queensland, Australia; the National Institute of Environmental Health
Sciences,||
Research Triangle Park, North
Carolina, and the Department of Medicine,**
University of North
Carolina at Chapel Hill, Chapel Hill, North Carolina
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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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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To establish the immunohistochemical KAI1 assay, we used the following formalin-fixed and paraffin-embedded cell lines as controls: rat prostate cancer cell line AT6.1,15 with and without the transfected KAI1 gene or transferred human chromosome 1116,17; human colon cancer cell lines SW480, SW620, RK0, and DLD-1; human primary rectal carcinoma cell lines SW837 and SW1463; and the androgen-independent human prostate cancer cell line PC-3. The AT6.1 cell line was a kind gift from Dr. J. Isaacs (Johns Hopkins University, Baltimore, MD). The human colorectal cancer cell lines were obtained from the American Type Tissue Collection (Manassas, VA). RKO was provided by Dr. Bert Vogelstein (Johns Hopkins University). The cell lines were grown in media recommended by the providers of the cell lines, in a humidified incubator with 5% CO2 at 37°C. All of the cell lines tested negative for Mycoplasma contamination at the National Institute of Environmental Health Sciences. In addition, we used several excess formalin-fixed, paraffin-embedded tissues from the Surgical Pathology Service at the University of North Carolina (UNC) Hospitals, including tonsil, colon, and breast. To study KAI1 expression in squamous and lymphoid neoplasms, we retrieved 18 archival head and neck squamous cell carcinomas (SqCCas) from the files of the UNC Surgical Pathology Service; 15 bronchogenic SqCCa from the Prince Charles Hospital in Queensland; 14 invasive and 10 intraepithelial squamous neoplasms of the cervix from the National Naval Medical Center in Bethesda, MD and from the Walter Reed Army Medical Center in Washington, DC; eight reactive lymph nodes, 13 follicular small cleaved (FSC), and 13 diffuse large cell (DLC) lymphomas from the files of the Armed Forces Institute of Pathology. The majority of these tumors had been fixed in formalin, although a subset of lymphoid lesions had been fixed in B-5. The mouse hybridoma cell line producing the anti-KAI1 antibody C33 was generously provided by Dr. Osamu Yoshie (Shionogi Institute for Medical Science, Osaka, Japan). The hybridoma supernatant was partially purified on a column. The stock antibody concentration of combined fractions 1 and 2 was 0.294 mg/ml. The immunohistochemical detection reaction utilized the Elite ABC kit from Vector (Burlingame, CA).
Western Analysis
The Western blot protocol used was based on a previously reported method.18 Cell monolayers were washed twice in cold phosphate-buffered saline (PBS). Then, 5 ml of cold PBS were added to the monolayer, and the cells were scraped into a 15-ml conical tube (Sarstedt, Newton, NC). The cells were centrifuged at 4°C for 5 minutes at 1200 rpm. The supernatant was aspirated, and the pellet was gently vortexed to briefly disperse the cells. Cell proteins were solubilized in 150–400 µl of lysis buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 3 mM MgCl2, and 0.5% NP-40, 2 mM phenylmethylsulfonyl fluoride) for 10 minutes on ice. The lysates were then centrifuged at 14,000 rpm at 4°C for 10 minutes. The supernatant was removed, and the protein concentration was determined by the Bradford method. An equal volume of Laemmli's sample buffer without 2-mercaptoethanol was added to the soluble protein and boiled for 5 minutes. The proteins were size fractionated by 17.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to Immobilon-P membrane (Millipore), and incubated with the KAI1 C33 hybridoma supernatant at a 1:100 dilution. Bound antibody was measured by the ECL Western blotting analysis system (Amersham Life Sciences, Buckinghamshire, England).
To ensure equal loading of protein, actin control experiments were performed as above, with the following modifications. Solubilized proteins were mixed with an equal volume of Laemmli's sample buffer supplemented with 10 mM dithiothreitol. The proteins were separated on a 10% SDS-PAGE gel, transferred to a nylon membrane, and incubated with an anti-actin monoclonal antibody (Sigma, St. Louis, MO).
Immunohistochemistry
Paraffin sections (4 µm) were placed on "Probe-on plus" slides (Fisher Scientific, Pittsburgh, PA), dewaxed in xylene, rehydrated, and heated in 0.01 M citrate buffer (pH 6) to 95–100°C in a steamer for 30 minutes. After washing in buffer, the slides were reacted with 0.2% trypsin (pH 7.8) for 3 minutes at room temperature (RT). After washing and quenching of endogenous peroxide, the sections were reacted with the C33 antibody (negative controls: nonspecific mouse IgG) overnight at RT. For cell blocks, the antibody was diluted 1:100; for tissues, the titer was 1:30. These conditions were determined after we tested several antigen retrieval methods, singly and in combination, a range of primary incubation times (1 hour to overnight), and a range of primary antibody concentrations (1:20 to 1:1000). The detection reaction followed the Vectastain recommended protocol (30-minute incubations). Several modifications to the latter, including a signal amplification step and use of streptavidin, did not improve the staining results. Diaminobenzidine was used for color development, and hematoxylin was used as the counterstain.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies indicated that KAI1 was expressed at high levels
in several cell types, including activated lymphocytes, and frozen
section immunohistochemistry (IHC) demonstrated that the antigen was
localized to the cell membrane.1-3,19
Thus, we used
archival tissues with abundant lymphoid cells, including tonsils, as
positive controls to develop an immunohistochemical assay for KAI1 in
paraffin sections. In addition, we utilized formalin-fixed and
paraffin-embedded cell lines that had defined KAI1 levels by Western
blot analysis (Table 1)
. Compared to
frozen tissues, paraffin sections required a higher concentration of
antibody and a longer incubation time to achieve comparable staining
results. The optimum titer was 1:100 for cell lines and 1:30 for
tissues. Moreover, a combination of two antigen retrieval methods,
heating in near-boiling citrate buffer and digestion with trypsin, was
required to detect the antigen (see Materials and Methods). We
determined the level of KAI1 protein expression by Western blotting in
10 cell lines (Figure 1A)
. There was a
good correlation between these data and the immunocytochemical staining
pattern of formalin-fixed, paraffin-embedded pellets of the same cell
lines (Table 1
, Figure 1
). In KAI1 negative cell lines such as AT6.1,
weak to moderate, nonspecific cytoplasmic reactivity, but no membrane
staining, was observed (Figure 1B)
. A culture of AT6.1 cells
transfected with a KAI1 expression vector showed strong
membrane reactivity and Golgi staining in the majority of the cells
(Figure 1C)
. Human rectal adenocarcinoma cells with a moderate to high
level of KAI1 (SW837) displayed distinct membrane staining in some, but
not all, cells (Figure 1D)
. Among the control tissues, human tonsil
consistently showed the strongest reactivity, with intense membrane
staining in both the squamous epithelium and the follicular centers
(Figure 2A)
. Because of the strong
reactivity of these two tissues, we proceeded to study the expression
of KAI1 in tumors derived from them. To assess KAI1 expression in a
lesion of interest, we always expected to see preserved reactivity in a
subset of admixed lymphocytes and/or macrophages or in nonneoplastic
squamous epithelium, when present.2,3,17-19
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Initially, we observed strong KAI1 staining in follicular
centers in several control tissues. We then studied lymphoid lesions
from a set of tumors that had previously been characterized for
abnormalities in the RB/p16 pathway (Table 2)
.20
All eight lymph nodes
with reactive changes were KAI1 positive. Ten of 13 FSC (77%) and
eight of 13 DLC (62%) lymphomas also showed distinct membrane
reactivity (Figure 2B)
. Conversely, three of 13 (23%) low-grade and
five of 13 (38%) high-grade lymphomas were negative for KAI1 (Figure 2C)
; there was preserved reactivity in benign lymphocytes and
macrophages.
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We studied 47 archival tumors from three different anatomic sites
(head and neck, lung, and cervix). Approximately 80% of the SqCCa
showed extensive loss of KAI1 staining, with relatively normal levels
in the remaining 20% (Table 3)
. In 26 of
37 tumors displaying KAI1 down-regulation, no definite membrane
staining was noted within the lesion. In 11 carcinomas, KAI1 expression
was confined to small nests of tumor cells (less than 10% of total).
In some cases, loss of KAI1 appeared to occur at the in situ
stage, with preserved membrane staining in adjacent dysplastic
epithelium (Figure 2D)
. Morphologically normal squamous epithelium,
when present, served as an internal positive control.
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To determine if loss of KAI1 may occur in preinvasive squamous neoplasia, as our observations in some tissues adjacent to invasive SqCCa suggested (see above), we evaluated 10 cases of cervical high-grade squamous intraepithelial lesion (HGSIL) that were not associated with invasive carcinoma. Seven lesions were diffusely and strongly positive, and two lesions were largely negative. In one case, there was strong staining superficially, but loss of reactivity in the deeper aspects. The latter staining pattern was unlikely to be artifactual, because inflammatory cells in the subjacent stroma stained positively.
Correlation of KAI1 Expression and Tumor Suppressor Gene Abnormalities
Because p53 purportedly activates KAI1 expression,21 we studied a group of lung cancers that had previously been analyzed for p53 abnormalities.22 In our limited number of cases (n = 15), there was no correlation between KAI1 down-regulation and aberrant p53 status, as determined by IHC, loss of heterozygosity (LOH) at 17p13, or single-strand conformational polymorphism (SSCP) abnormalities in exons 5–8 (data not shown). The downstream signaling pathways for KAI1 are unknown; therefore we were interested in the relationship between KAI1 expression and p16 and pRB, two gene products that are critical in the regulation of cell cycle progression. The same group of lung cancers, along with series of head and neck cancers and lymphomas, had previously been characterized for the expression patterns of p16 and pRB (n = 57).20,22 We did not find an association between the absence of p16 or pRB and down-regulation of KAI1 (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several methodological approaches have been described to determine the level of KAI1 expression in human tissues, including RT-PCR and Northern and Western analysis.9,10,16 Interpretation of such analyses is rendered difficult by the invariable presence of nonneoplastic cells that may express KAI1 at high levels. To circumvent this problem, other investigators have employed in situ hybridization5,25 or immunohistochemistry.2,3,13,26 The obvious advantage of these techniques is that they allow the evaluation of KAI1 expression specifically in the cell population(s) of interest.
The anti-KAI1 antibody we used, C33, is among several antibodies that have been used successfully for analysis of KAI1 by frozen section immunohistochemistry.18,19 Strong membrane staining in benign prostatic epithelial cells and down-regulation in a large proportion of prostatic adenocarcinomas were described by two groups.1,26 Huang et al found a high frequency of reduced KAI1 expression in carcinomas of the lung, breast, colon, and stomach.3 However, several laboratories reported difficulties with the immunohistochemical analysis of archival tissues.1,3 We are aware of very few previous studies that analyzed KAI1 levels in paraffin-embedded tumors. In one series, there was loss of KAI1 expression in some archival prostatic adenomacarcinomas, but about half of the cases were nonreactive, and the tissues were significantly less reactive compared to frozen sections.17 In a small series of 14 paraffin-embedded colonic adenocarcinomas, down-regulation of KAI1 was found in a subset of them.6 Friess et al found a correlation of KAI1 expression by IHC, ISH, and Northern blotting in pancreatic carcinomas.25
An important goal of our study was to establish a reliable
immunohistochemical assay for KAI1 in paraffin-embedded tissues, which
would enable us to correlate KAI1 levels with the metastatic phenotype
and other biological and clinical features of archival tumors. We are
confident that our assay has adequate specificity, because cell lines
known to lack KAI1 did not show the expected staining pattern (Table 1
,
Figure 1B
). When KAI1 was expressed, it was associated with a specific
membrane staining pattern (Figure 1C,D)
. In cell lines transfected
with KAI1 or transferred with chromosome 11,
immunocytochemistry showed the presence of expressed protein in a
subset of the cells (Figure 1C)
. Interestingly, in KAI1-positive cell
lines, not all cells showed membrane reactivity, and furthermore, the
staining intensity was variable (Figure 1D)
, suggesting that expression
of the glycoprotein may be subject to yet unknown regulatory
mechanisms. Overall, there was excellent qualitative agreement between
protein expression determined by Western analysis and by
immunocytochemistry (Table 1)
. Like other investigators, we noted that
fixation and processing greatly reduced KAI1 antigenicity, which could
be partially restored with rather stringent antigen retrieval methods.
The combination of techniques described here has not been reported
previously. An adverse side effect of this treatment is the partial
destruction of some tissue sections, which may require restaining or
use of alternative paraffin blocks.
Although we could convincingly demonstrate the marked down-regulation of KAI1 in lymphoid and squamous neoplasms (see below), we cannot rule out the possibility that low levels of this glycoprotein still exist in the tumor cells. The mechanism of KAI1 down-regulation is not known. The 5' promoter region of the gene contains a CpG island,27 raising the possibility of gene silencing by promoter methylation. Mutations in the gene appear to be uncommon,1 and it is unclear how they might affect the immunohistochemical reactivity of the protein. The KAI1 gene is located on chromosome 11p11.2.8,16 Although allelic loss at this site may be observed in prostate cancer,8,16 there is no mutation of the remaining allele. Whatever the mechanism, IHC is a suitable modality for demonstrating the relative or absolute lack of KAI1 in neoplastic tissues. One additional limitation of this technique, however, is that it is unlikely to detect aberrant KAI1 glycosylation. The latter has been observed in several types of tumors, although the functional significance of this type of posttranslational modification remains unclear.9,18,28 However, it was previously demonstrated that highly branched asparagine- (N-) linked glycosylation of glycoproteins was correlated with the malignant phenotype in rodent and human models.29
During the establishment of the immunohistochemical KAI1 assay, we
noted that strong membrane staining was present in germinal centers.
This staining pattern was previously described by others17
and is consistent with earlier reports of high KAI1 levels in activated
lymphocytes.18,19,30
To test the hypothesis that KAI1
down-regulation may occur in lymphoid neoplasia, we stained eight
reactive lymph nodes as well as 13 FSC (low-grade) and 13 DLC
(high-grade) lymphomas. As expected, all benign lymph nodes were KAI1
positive. In contrast, about one-quarter of FSC and over one third of
DLC lymphomas were KAI1 negative, with preserved staining in admixed
nonneoplastic cells (Table 2
, Figure 2C
). Whether loss of KAI1 in
lymphomas is associated with a more aggressive clinical phenotype
remains to be determined.
We also found strong membrane staining in squamous epithelia,
consistent with previous reports.2
We observed extensive
down-regulation in 37 of 47 squamous cell carcinomas from the lung,
head and neck, and cervix, and in the majority of down-regulated cases
there was no membrane staining in the invasive portion of the tumor
(Figure 2D)
. In an IHC study on skin frozen sections, Okochi et al
reported marked down-regulation of KAI1 in basal cell
carcinomas.2
Because the latter rarely metastasize, it was
suggested that KAI1 loss may be important for stromal invasion in this
tumor type. Our data support the hypothesis that down-regulation of
KAI1 may be an important event in the progression of squamous cell
carcinomas from the in situ to the invasive stage. There is
previous immunohistochemical evidence that KAI1 may be reduced or
absent in up to two-thirds of squamous cell carcinomas of the
lung,14
which correlated well with an earlier study using
the RT-PCR technique.11
Those data are in agreement with
our observation that KAI1 was down-regulated in 11 of 15 (73%)
bronchogenic SqCCa. Finally, White et al reported that 10 of 11
non-small-cell lung cancer cell lines were KAI1 negative.28
There are few data on KAI1 expression in preinvasive neoplasia. Okochi et al did not find evidence of loss of KAI1 expression in cutaneous squamous cell carcinomas in situ.2 We observed KAI1 down-regulation in some precursor lesions associated with invasive squamous cell carcinomas. We studied 10 additional high-grade dysplasias from the cervix, which showed several staining patterns. Seven were diffusely positive, two were largely negative, and one case displayed loss of KAI1 staining at the base of the lesion. Therefore, KAI1 down-regulation can occur in preinvasive squamous neoplasms, but it is unknown whether it increases the likelihood of stromal invasion. Larger studies with clinical follow-up data are required to address this issue.
It was recently suggested that expression of KAI1 is controlled, in part, by p53.21 Among the lesions we studied, only the small number of bronchogenic SqCCa had previously been tested for p53 abnormalities. No correlation between the latter and KAI1 expression was found, but the statistical power was limited because of small numbers. As far as we know, the relationship between KAI1 status and cell cycle regulation has not been studied. We had previously determined the presence or absence of two important cell cycle regulatory proteins, p16 and pRB, in 57 of the 73 tumors included in this study. We found no correlation between KAI1 and p16 or pRB expression. This observation is of some interest because a significant subset of tumors show p16 inactivation by promoter hypermethylation.31 KAI1 contains CpG islands in the 5' portion of the gene,27 and it is tempting to speculate that methylation of the promoter may be a mode of down-regulation of this gene as well.
In conclusion, we describe a method for detecting aberrant KAI1 expression in archival tissues by immunohistochemistry. Although markedly reduced KAI1 reactivity is considered abnormal, a normal staining pattern does not necessarily imply a normally functioning gene product. The technique may not be suitable for detecting abnormal glycosylation patterns that may be functionally important in some tumors.9,18 Loss of KAI1 occurred in a significant subset of lymphomas. The frequent down-regulation of the gene in squamous cell carcinomas, and its loss in some preinvasive squamous neoplasms suggest an important role of KAI1 in the progression to the invasive phenotype. It is hoped that the technique described here may facilitate studies on archival tissues that further define the role of KAI1 in the development of human tumors and their metastases.
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Acknowledgements |
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Footnotes |
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D.P.L. was supported by a grant from the National Institutes of Health (MO1 RR00046-38S2).
Accepted for publication March 12, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3ß1 integrin. Br J Dermatol 1997, 137:856-863[Medline]
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