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Abnormalities in retinoic acid receptor beta (RAR-β) are common in human lung cancer
1
II, sialyllacto-N-fucopentaose III, disialyllacto-N-tetraose A-heptasaccharide, and Bi-antennarv octasaccharide. Several antibodies detect unique carbohydrate epitopes which may be useful as tumor markers for lung cancer. EXPRESSION OF THE PROTEIN KINASE P-66 RECOGNIZED BY THE HONOCLONAL ANTIBODY TJ4C4 IN HUMAN LUNG %;PLASHS. GK H(81:es,’ G Ghadge,’ 9 Thimmappaya’ dA Radosevlch DeDts. of ‘Pathology, ‘Hlcroblology/Immunology and ‘Medicine. Northwestern University Medical School/VA Lakeslde Medical Center, Chtcago, U.S.A. nhlch P68 IS an interferon rnducible protein k1nas.e 1s belreved to be a” important factor I” the regulation of both viral and cellular protein synthesis. We have PreviOuely reported on a monoclonal antibody (TJ4C4) whlCh speclflcally detects ~66, and which can be used to detect th16 ;::;;:n 1” formal,” fixed. paraffin embedded Because of Its important role in cellular protein synthesis, we hypothesized that ~66 expression would vary among lung neoplasms as a reflection of their diverse blologlc nature. A total of 246 untreated prrmary pulmonary and pleural neoplasms were Studled. relative intensity The frequency and Of ~68 expreSsion was determined by light microscopic evaluation of AQC 1mmunoperoXldase stained specimens. All categor,es of tumors studled demonstrated a spectrum of p6S expresslo”. Higher levels of ~68 expression were seen in grade 1 squamous cell carcinoma (SSC) and adenocsrcinoma (AC) than in their more poorly differentiated counterparts. Expression Of the antigen in undifferentiated large cell carcinoma Has Similar to that seen in grades 2 and 3 AC and SW. Sronchioalveolar carcinoma (BAC) expressed very low levels of ~68 despite Its well differentiated appearance. This marked contrast further distinguishes BAC from AC. Nouroendccrine tumors showed dlfferenfial ~66 expresslon With the lntermediats variety of small cell carcinoma expressing higher levels of ~68 than the classic “oat cell” form. HesotheliOmas weakly expressed ~66, ulth relatively higher levels I” the epitheliold than in the Spindle cell varieties. Dlfferentlal express~o” of p66 by lung and pleural neoplasms Points to biologic differences between these tumors, and may provide clinically relevant informatlon about individual tumor subtypes. Extracellular Matrix in Preneo lastic Lesions and Early Cancer of B. Voss and K.-M. Bochum, Germany Characterization of preneoplastic lesions mainly concentrated on cellular, nuclear and epithelial atypias. The extracellular matrix was almost neglected, although malignant turnour cells interact with exrracellular matrix molecules during tumour invasion. Until now systematic investigations of extracellular matrix corn onents in preneo P lastic lesions and early stages of lung cancer are ackmg. P 1 0 preneoplastic lesions, 10 specimens of early cancer and 30 specimens of normal bronchial mucosa were examined by means of immunofluorescence microscopy. The distribution of collagen type I and III and the non-collagenous glycoproteins laminin and fibronectin have been investigated by an indirect immunohisto- chemical method. With an increasing degree of preneoplasia an increased matrix disarrangement of the basement mebrane zone could be observed. In severe dysplasia and carcinoma in situ neosynthesis of collagen type III and especially laminin in close association to neoangiogenesis could be demonstrated besides a disintegration of extracellular matrix components. In early lung cancer numerous laminin positive basement membrane-like structures are situated around turnour cells. An enhanced deposition of collagen type I and III fibres could be demonstrated around tumour cell Islets. The results indicate a partial loss of function in preneoplastic basal cells in cases of dysplasia and carcinoma in situ. In early stages of invasive squambus cell carcinoma of the bronchus, extracellular matrix comoonents obviouslv could be oroduced bv tumour cells. Functional Expression of Specific Subsets of Enhancer Binding Proteins Associated with the Differentiation State of Lung Carcinomas. Yuk-Chor Wong and Samuel D. Bernal Section of Medical Oncology, University Hospital, Roston, MA 02118. Enhancersareimportantelementsin cell-typespecificregulation of gene expression and differentiation. However, enhancer cell-type specificity is determined by the availability of s trans-acting enhancer binding proteins in that particular ceP ecific Based on the assum I type. ferent subsets of en 1 tion that different cell-types contain dif- ancer binding proteins, we developed a functional assay of heterologous enhancers and their corresponding bmding proteins to characterize the differenti- ation state of various morphological types of lung carcinoma cell lines. Using a reporter gene codmg for chloramphenicol acetyltransferase,non-small-cellcarcinomaswerefound tobe2-3 orders of magnitude more efficient in utilizin 8 the SV40 enhancer. Using specific enhancer sequences, specl XC subsets of cellular enhancer-binding proteins were found in the different morphologic types of lung cancers. Nuclear extracts from ade- nocarcinomas and squamous carcinomaswere found lo have high binding activities to NF-I, AP-1, AP-2 and AP-3 consensus sequences, compared to large-cell and small-cell carcinomas. Adenocarcinoma formed specific NF-I DNA-protein comolexes which were different fromsquamous carcinoina. Whereas clas- sical small cell carcinoma contained very small amounts of NFl DNA-orotein comolexes. variant small cell carcinoma formed larger’amounts of’these complexes and showed a pattern that resembledthe NFI DNA-proteincomplexesofadenocarcinomas but not squamous carcinomas. Our results su ,pt of enhancers can be used to characterize the cl1 tbat,a panel erentlatlon state of lung carcinomas. Information obtained from such analysis may be useful in the understanding of the differentiation programs in normal and neoplastic lung epithelium. ABNORMALITIES IN RETINOIC ACID RECEPTOR BETA (RAR-b) ARE COMMON IN HUFAN LUNG CqNCER Benjamin G V. Fragionil, Neell, Johannes F. Gebyt , Nadeem Moghul , John and David Sugarbaker 1Molecular Medicine Unit, Beth Israel Hospital, Boston, MA and *Dept. of Thoracic Surgery, Brigham and Women’s Hospital, Boston, MA. Virtually all lung tumors, both SCLC and NSCLC, have deletions on the short arm of chromosome 3. Although the precise boundaries of these deletions are unclear. most evidence sueeests that a tumor suppressor gene(s) is locateh in the 3~21-24 r&$on. Since retinoids are known to modulate epithelial cell growth and differentiation, and vitamin A deficiency causes squamous metaplasia of bronchial epithelium, a studied RAR-6 exuression in twentv-four une cancer cells lines and P remalignant lesion, we nine ,R rimary iumbrs. In normal t;acheobroi&hial epithelium and perlp era1 lung, RAR-lis expressed as two mRNAs of sizes 3.2 and 2.8 kb. About 25% of lung cancer cell lines lack detectable RAR-P expression, one has substantially reduced amounts of a truncated transcript, ten to fifteen percent showed selective loss of the lower mRNA and the remainder showed the normal doublet E attern. In some cases, expression of one or both transcripts could e induced bv treatment with oharmacoloeic doses of retinoic acid. but in others, the RAR-P geie was com$etely silent. Absent 0; significantly decreased RAR-6 RNA was also found in 3 of the primaly tuinors and another iumor showed selective loss of the lower transcriot. Southern analvsis of 14 of the cell lines revealed in at least one allele of RAR-0 in three. sequencing is in progress to determine whether more subtle abnormalities exist in those samples that express apparently normal RAR-B transcripts. Although we have found examples of RAR+ abnormalities in all histologic subtypes of lung cancer, squamous cell carcinomas seem to be consistently abnormal. Our data su est that abnormalities in the structure and expression of RAR-b pYg ay an Important role in the pathogenesis of human lung cancer. RAR_8 may be a new type of tumor suppressor gene, which functions to maintain normal cytodifferentiation.
Transcript of Abnormalities in retinoic acid receptor beta (RAR-β) are common in human lung cancer
II, sialyllacto-N-fucopentaose III, disialyllacto-N-tetraose A-heptasaccharide, and Bi-antennarv octasaccharide. Several antibodies detect unique carbohydrate epitopes which may be useful as tumor markers for lung cancer.
EXPRESSION OF THE PROTEIN KINASE P-66 RECOGNIZED BY THE HONOCLONAL ANTIBODY TJ4C4 IN HUMAN LUNG %;PLASHS. GK H(81:es,’ G Ghadge,’ 9 Thimmappaya’
dA Radosevlch DeDts. of ‘Pathology, ‘Hlcroblology/Immunology and ‘Medicine. Northwestern University Medical School/VA Lakeslde Medical Center, Chtcago, U.S.A.
nhlch P68 IS an interferon rnducible protein k1nas.e
1s belreved to be a” important factor I” the regulation of both viral and cellular protein synthesis. We have PreviOuely reported on a monoclonal antibody (TJ4C4) whlCh speclflcally detects ~66, and which can be used to detect th16 ;::;;:n 1” formal,” fixed. paraffin embedded
Because of Its important role in cellular protein synthesis, we hypothesized that ~66 expression would vary among lung neoplasms as a reflection of their diverse blologlc nature. A total of 246 untreated prrmary pulmonary and pleural neoplasms were Studled. relative intensity
The frequency and Of ~68 expreSsion was
determined by light microscopic evaluation of AQC 1mmunoperoXldase stained specimens. All categor,es of tumors studled demonstrated a spectrum of p6S expresslo”. Higher levels of ~68 expression were seen in grade 1 squamous cell carcinoma (SSC) and adenocsrcinoma (AC) than in their more poorly differentiated counterparts. Expression Of the antigen in undifferentiated large cell carcinoma Has Similar to that seen in grades 2 and 3 AC and SW. Sronchioalveolar carcinoma (BAC) expressed very low levels of ~68 despite Its well differentiated appearance. This marked contrast further distinguishes BAC from AC. Nouroendccrine tumors showed dlfferenfial ~66 expresslon With the lntermediats variety of small cell carcinoma expressing higher levels of ~68 than the classic “oat cell” form. HesotheliOmas weakly expressed ~66, ulth relatively higher levels I” the epitheliold than in the Spindle cell varieties. Dlfferentlal express~o” of p66 by lung and pleural neoplasms Points to biologic differences between these tumors, and may provide clinically relevant informatlon about individual tumor subtypes.
Extracellular Matrix in Preneo lastic Lesions and Early Cancer of B. Voss and K.-M.
Bochum, Germany Characterization of preneoplastic lesions mainly concentrated
on cellular, nuclear and epithelial atypias. The extracellular matrix was almost neglected, although malignant turnour cells interact with exrracellular matrix molecules during tumour invasion. Until now systematic investigations of extracellular matrix corn onents in preneo
P lastic lesions and early stages of lung cancer are ackmg. P
1 0 preneoplastic lesions, 10 specimens of early cancer and 30 specimens of normal bronchial mucosa were examined by means of immunofluorescence microscopy. The distribution of collagen type I and III and the non-collagenous glycoproteins laminin and fibronectin have been investigated by an indirect immunohisto- chemical method. With an increasing degree of preneoplasia an increased matrix disarrangement of the basement mebrane zone could be observed.
In severe dysplasia and carcinoma in situ neosynthesis of collagen type III and especially laminin in close association to neoangiogenesis could be demonstrated besides a disintegration of extracellular matrix components. In early lung cancer numerous laminin positive basement membrane-like structures are situated around turnour cells. An enhanced deposition of collagen type I and III fibres could be demonstrated around tumour cell Islets.
The results indicate a partial loss of function in preneoplastic basal cells in cases of dysplasia and carcinoma in situ. In early stages of invasive squambus cell carcinoma of the bronchus, extracellular matrix comoonents obviouslv could be oroduced bv tumour cells.
Functional Expression of Specific Subsets of Enhancer Binding Proteins Associated with the Differentiation State of Lung Carcinomas.
Yuk-Chor Wong and Samuel D. Bernal
Section of Medical Oncology, University Hospital, Roston, MA 02118. Enhancersareimportantelementsin cell-typespecificregulation of gene expression and differentiation. However, enhancer cell-type specificity is determined by the availability of s trans-acting enhancer binding proteins in that particular ce P
ecific
Based on the assum I type.
ferent subsets of en 1 tion that different cell-types contain dif- ancer binding proteins, we developed a
functional assay of heterologous enhancers and their corresponding bmding proteins to characterize the differenti- ation state of various morphological types of lung carcinoma cell lines. Using a reporter gene codmg for chloramphenicol acetyltransferase,non-small-cellcarcinomaswerefound tobe2-3 orders of magnitude more efficient in utilizin
8 the SV40
enhancer. Using specific enhancer sequences, specl XC subsets of cellular enhancer-binding proteins were found in the different morphologic types of lung cancers. Nuclear extracts from ade- nocarcinomas and squamous carcinomaswere found lo have high binding activities to NF-I, AP-1, AP-2 and AP-3 consensus sequences, compared to large-cell and small-cell carcinomas. Adenocarcinoma formed specific NF-I DNA-protein comolexes which were different fromsquamous carcinoina. Whereas clas- sical small cell carcinoma contained very small amounts of NFl DNA-orotein comolexes. variant small cell carcinoma formed larger’amounts of’these complexes and showed a pattern that resembledthe NFI DNA-proteincomplexesofadenocarcinomas but not squamous carcinomas. Our results su ,pt of enhancers can be used to characterize the cl1
tbat,a panel erentlatlon state
of lung carcinomas. Information obtained from such analysis may be useful in the understanding of the differentiation programs in normal and neoplastic lung epithelium.
ABNORMALITIES IN RETINOIC ACID RECEPTOR BETA (RAR-b) ARE COMMON IN HUFAN LUNG CqNCER Benjamin G V. Fragionil,
Neell, Johannes F. Gebyt , Nadeem Moghul , John and David Sugarbaker 1Molecular Medicine Unit,
Beth Israel Hospital, Boston, MA and *Dept. of Thoracic Surgery, Brigham and Women’s Hospital, Boston, MA. Virtually all lung tumors, both SCLC and NSCLC, have deletions on the short arm of chromosome 3. Although the precise boundaries of these deletions are unclear. most evidence sueeests that a tumor suppressor gene(s) is locateh in the 3~21-24 r&$on. Since retinoids are known to modulate epithelial cell growth and differentiation, and vitamin A deficiency causes squamous metaplasia of bronchial epithelium, a studied RAR-6 exuression in twentv-four une cancer cells lines and P
remalignant lesion, we
nine ,R
rimary iumbrs. In normal t;acheobroi&hial epithelium and perlp era1 lung, RAR-lis expressed as two mRNAs of sizes 3.2 and 2.8 kb. About 25% of lung cancer cell lines lack detectable RAR-P expression, one has substantially reduced amounts of a truncated transcript, ten to fifteen percent showed selective loss of the lower mRNA and the remainder showed the normal doublet
E attern. In some cases, expression of one or both transcripts could e induced bv treatment with oharmacoloeic doses of retinoic acid.
but in others, the RAR-P geie was com$etely silent. Absent 0; significantly decreased RAR-6 RNA was also found in 3 of the primaly tuinors and another iumor showed selective loss of the lower transcriot. Southern analvsis of 14 of the cell lines revealed
in at least one allele of RAR-0 in three. sequencing is in progress to determine
whether more subtle abnormalities exist in those samples that express apparently normal RAR-B transcripts. Although we have found examples of RAR+ abnormalities in all histologic subtypes of lung cancer, squamous cell carcinomas seem to be consistently abnormal. Our data su est that abnormalities in the structure and expression of RAR-b p Yg ay an Important role in the pathogenesis of human lung cancer. RAR_8 may be a new type of tumor suppressor gene, which functions to maintain normal cytodifferentiation.
