Tetraspanin protein KAI1 expression in retinoblastoma
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《英国眼科学杂志》
1 Department of Ocular Pathology, Medical and Vision Research Foundation, Sankara Nethralaya, Chennai, India
2 Department of Ocular Oncology, Medical and Vision Research Foundation, Sankara Nethralaya, Chennai, India
Correspondence to:
Dr Subramanian Krishnakumar
Vision Research Foundation, Sankara Nethralaya, 18, College Road, Chennai 600 006, Tamil Nadu, India; drkrishnakumar_2000@yahoo.com
Accepted for publication 1 August 2003
Keywords: KAI1; retinoblastoma; immunohistochemistry; tetraspanin
KAI1/CD82 is a metastasis suppressor gene located on human chromosome 11p11.2. It is a member of the structurally distinct family of cell surface glycoprotein, transmembrane 4-protein superfamily.1 KAI1 was initially isolated as a gene that suppressed metastasis of rat prostate tumour cells.2 KAI1 is downregulated in several types of human malignancies.3–5 The purpose of this study was to investigate the expression of KAI1 in retinoblastoma and to correlate clinicopathologically.
Methods
There were 30 archival specimens of retinoblastomas from 2000 to 2002. There were 12 tumours with no invasion of choroid, or optic nerve and 18 tumours with invasion of choroid/optic nerve, and one tumour which had metastasised to the submandibular region. There were six well differentiated tumours, six moderately differentiated tumours, and 18 poorly differentiated tumours (table 1). Immunohistochemical staining was performed using a sensitive labelled streptavidin biotin (LSAB kit, Dako) on tumours using monoclonal antibodies for tetraspanin KAI1 (C33, Novacastra) and for proliferation index Ki-67 (Clone MIB-1, Dako, Denmark) after antigen retrieval.
Table 1 Clinicopathological and immunohistochemical features of the retinoblastomas used in the study
The immunoanalysis for KAI1 was done based on the percentage of cells and the staining intensity. KAI1 was scored after analysing 15 tumour fields and on a four tiered scale: 0, negative (0%–5% positive cells); 1+, weak to moderate (6%–30%); 2+, intense (31%–50%); and 3+, very intense (>50%). Labelling index for Ki 67 positive cells was expressed by counting a minimum of 500 cells in the staining area. Tumours were divided into two groups; group I >50% of cells showing positive Ki 67 expression and group II <50% of cells showing positive for Ki 67. Data were analysed for statistical significance.
Results
The immunohistochemical details are given in table 1. KAI1 expression was seen in the control lymphoid follicle of the tonsil (fig 1A). Intense KAI1 positivity with more than 80% positivity was seen in all 12/12 tumours with no invasion (fig 1B). Among the 18/18 tumours with invasion, KAI1 was decreased in all 18. The invading front of the tumour had less KAI1 than the tumour at the central portion. Retinoblastomas with focal and diffuse invasion of choroid had negative KAI1 immunoreactivity. Tumours with prelaminar optic nerve invasion had weak KAI1 immunoreactivity (fig 1C). Tumours at the post-laminar and surgical end of the optic nerve (inset, fig 1C) and at the metastatic site (fig 1D) had negative KAI1 immunoreactivity. Negative KAi1 reactivity was seen in 50% (9/18) of poorly differentiated retinoblastomas. Statistically significant correlation was observed between KAi1 expression and Ki 67 labelling index in the whole study group (p<0.001). No statistically significant correlation was observed between KAI1 expression and differentiation. Significant statistical correlation was observed when KAI1 expression was compared with tumour invasion (p<0.001).
Figure 1 (A) Microphotograph showing the KAI1 expression in control lymphoid follicle of the tonsil (magnification x200). (B) Microphotograph showing the intense membrane immunoreactivity of KAI1 in retinoblastoma with no invasion (magnificationx200). (C) Microphotograph showing the weak KAI1 immunoreactivity of tumour cells invading the lamina cribrosa of the optic nerve (magnification x100). Inset shows negative KAI1 in tumour cells at the surgical end of optic nerve. (D) Microphotograph showing the negative KAI1 immunoreactivity of tumour cells at the metastatic site in the submandibular region (magnificationx200).
Comment
Retinoblastoma joins a growing list of cancers in which downregulation of KAI1 is associated with tumour progression. In our study KAI1 was identified by the monoclonal antibody CD33.6 It was originally shown as inhibitory to syncytium formation induced by human T cell leukaemia virus type I, and this specific inhibition to syncytium formation induced by some human T cell line by this antibody was strongly associated with altered glycosylation of cell surface antigen, suggesting that the C33 antigen—that is, KAI1, might have a possible role in the cell to cell adhesion mechanism.7
Thus, KAI1 may link to the cell surface molecules, such as integrins, E-cadherin, and other TM4SF members, and loss of KAI1 function may have a significant role in the progression of retinoblastoma.7 The mechanism of KAI1 downregulation is not known. The 5’ promoter region of the gene contains a CpG island,8 raising the possibility of gene silencing by promoter methylation. Thus, biologically, our findings suggest a potential implication of KAI1 in tumour progression and these molecules may provide novel insights into tumour progression in retinoblastoma.
ACKNOWLEDGEMENTS
Financial support: Indian Council Of Medical Research, India.
References
Maecker HT, Todd SC, Levy S. The tetraspanin superfamily: molecular facilitators. Faseb J 1997;11:428–42.
Dong JT, Lamb PW, Rinker-Schaeffer CW, et al. Kai1, a metastasis suppressor gene for prostate cancer on human chromosome 11p11. 2. Science (Washington DC) 1995;268:884–6.
Yu Y, Yang J-L, Markovic B, et al. Loss of KAI1 messenger RNA expression in both high-grade and invasive human bladder cancers. Clin Cancer Res 1997;3:1045–9.
Huang CI, Kohno N, Ogawa E, et al. Correlation of reduction in mrp-1/cd9 and KAI1/cd82 expression with recurrences in breast cancer patients. Am J Pathol 1998;153:973–83.
Guo X, Friess H, Graber HU, et al. KAI1 expression is up-regulated in early pancreatic cancer and decreased in the presence of metastases. Cancer Res 1996;56:4876–80.
Imai T, Fukudome K, Takagi S, et al. C33 antigen recognized by monoclonal antibodies inhibitory to human T cell leukemia virus type 1-induced syncytium formation is a member of a new family of transmembrane proteins including cd9, cd37, cd53, and cd63. J Immunol 1992;149:2879–86.
Rubinstein E, Boucheix C. Tetraspans. In: Kreis T, Vale R, eds. Guidebook to the extracellular matrix and adhesion proteins. Oxford: Oxford University Press, 1999:321–4.
Dong JT, Isaacs WB, Barrett JC, et al. Genomic organization of the human KAI1 metastasis-suppressor gene. Genomics 1997;41:25–32.(S AmirthaLakshmi1, V Push)
2 Department of Ocular Oncology, Medical and Vision Research Foundation, Sankara Nethralaya, Chennai, India
Correspondence to:
Dr Subramanian Krishnakumar
Vision Research Foundation, Sankara Nethralaya, 18, College Road, Chennai 600 006, Tamil Nadu, India; drkrishnakumar_2000@yahoo.com
Accepted for publication 1 August 2003
Keywords: KAI1; retinoblastoma; immunohistochemistry; tetraspanin
KAI1/CD82 is a metastasis suppressor gene located on human chromosome 11p11.2. It is a member of the structurally distinct family of cell surface glycoprotein, transmembrane 4-protein superfamily.1 KAI1 was initially isolated as a gene that suppressed metastasis of rat prostate tumour cells.2 KAI1 is downregulated in several types of human malignancies.3–5 The purpose of this study was to investigate the expression of KAI1 in retinoblastoma and to correlate clinicopathologically.
Methods
There were 30 archival specimens of retinoblastomas from 2000 to 2002. There were 12 tumours with no invasion of choroid, or optic nerve and 18 tumours with invasion of choroid/optic nerve, and one tumour which had metastasised to the submandibular region. There were six well differentiated tumours, six moderately differentiated tumours, and 18 poorly differentiated tumours (table 1). Immunohistochemical staining was performed using a sensitive labelled streptavidin biotin (LSAB kit, Dako) on tumours using monoclonal antibodies for tetraspanin KAI1 (C33, Novacastra) and for proliferation index Ki-67 (Clone MIB-1, Dako, Denmark) after antigen retrieval.
Table 1 Clinicopathological and immunohistochemical features of the retinoblastomas used in the study
The immunoanalysis for KAI1 was done based on the percentage of cells and the staining intensity. KAI1 was scored after analysing 15 tumour fields and on a four tiered scale: 0, negative (0%–5% positive cells); 1+, weak to moderate (6%–30%); 2+, intense (31%–50%); and 3+, very intense (>50%). Labelling index for Ki 67 positive cells was expressed by counting a minimum of 500 cells in the staining area. Tumours were divided into two groups; group I >50% of cells showing positive Ki 67 expression and group II <50% of cells showing positive for Ki 67. Data were analysed for statistical significance.
Results
The immunohistochemical details are given in table 1. KAI1 expression was seen in the control lymphoid follicle of the tonsil (fig 1A). Intense KAI1 positivity with more than 80% positivity was seen in all 12/12 tumours with no invasion (fig 1B). Among the 18/18 tumours with invasion, KAI1 was decreased in all 18. The invading front of the tumour had less KAI1 than the tumour at the central portion. Retinoblastomas with focal and diffuse invasion of choroid had negative KAI1 immunoreactivity. Tumours with prelaminar optic nerve invasion had weak KAI1 immunoreactivity (fig 1C). Tumours at the post-laminar and surgical end of the optic nerve (inset, fig 1C) and at the metastatic site (fig 1D) had negative KAI1 immunoreactivity. Negative KAi1 reactivity was seen in 50% (9/18) of poorly differentiated retinoblastomas. Statistically significant correlation was observed between KAi1 expression and Ki 67 labelling index in the whole study group (p<0.001). No statistically significant correlation was observed between KAI1 expression and differentiation. Significant statistical correlation was observed when KAI1 expression was compared with tumour invasion (p<0.001).
Figure 1 (A) Microphotograph showing the KAI1 expression in control lymphoid follicle of the tonsil (magnification x200). (B) Microphotograph showing the intense membrane immunoreactivity of KAI1 in retinoblastoma with no invasion (magnificationx200). (C) Microphotograph showing the weak KAI1 immunoreactivity of tumour cells invading the lamina cribrosa of the optic nerve (magnification x100). Inset shows negative KAI1 in tumour cells at the surgical end of optic nerve. (D) Microphotograph showing the negative KAI1 immunoreactivity of tumour cells at the metastatic site in the submandibular region (magnificationx200).
Comment
Retinoblastoma joins a growing list of cancers in which downregulation of KAI1 is associated with tumour progression. In our study KAI1 was identified by the monoclonal antibody CD33.6 It was originally shown as inhibitory to syncytium formation induced by human T cell leukaemia virus type I, and this specific inhibition to syncytium formation induced by some human T cell line by this antibody was strongly associated with altered glycosylation of cell surface antigen, suggesting that the C33 antigen—that is, KAI1, might have a possible role in the cell to cell adhesion mechanism.7
Thus, KAI1 may link to the cell surface molecules, such as integrins, E-cadherin, and other TM4SF members, and loss of KAI1 function may have a significant role in the progression of retinoblastoma.7 The mechanism of KAI1 downregulation is not known. The 5’ promoter region of the gene contains a CpG island,8 raising the possibility of gene silencing by promoter methylation. Thus, biologically, our findings suggest a potential implication of KAI1 in tumour progression and these molecules may provide novel insights into tumour progression in retinoblastoma.
ACKNOWLEDGEMENTS
Financial support: Indian Council Of Medical Research, India.
References
Maecker HT, Todd SC, Levy S. The tetraspanin superfamily: molecular facilitators. Faseb J 1997;11:428–42.
Dong JT, Lamb PW, Rinker-Schaeffer CW, et al. Kai1, a metastasis suppressor gene for prostate cancer on human chromosome 11p11. 2. Science (Washington DC) 1995;268:884–6.
Yu Y, Yang J-L, Markovic B, et al. Loss of KAI1 messenger RNA expression in both high-grade and invasive human bladder cancers. Clin Cancer Res 1997;3:1045–9.
Huang CI, Kohno N, Ogawa E, et al. Correlation of reduction in mrp-1/cd9 and KAI1/cd82 expression with recurrences in breast cancer patients. Am J Pathol 1998;153:973–83.
Guo X, Friess H, Graber HU, et al. KAI1 expression is up-regulated in early pancreatic cancer and decreased in the presence of metastases. Cancer Res 1996;56:4876–80.
Imai T, Fukudome K, Takagi S, et al. C33 antigen recognized by monoclonal antibodies inhibitory to human T cell leukemia virus type 1-induced syncytium formation is a member of a new family of transmembrane proteins including cd9, cd37, cd53, and cd63. J Immunol 1992;149:2879–86.
Rubinstein E, Boucheix C. Tetraspans. In: Kreis T, Vale R, eds. Guidebook to the extracellular matrix and adhesion proteins. Oxford: Oxford University Press, 1999:321–4.
Dong JT, Isaacs WB, Barrett JC, et al. Genomic organization of the human KAI1 metastasis-suppressor gene. Genomics 1997;41:25–32.(S AmirthaLakshmi1, V Push)