Two cell adhesion molecules, nectin and cadherin, interact through their cytoplasmic domain-associated proteins.

We have found a new cell-cell adhesion system at cadherin-based cell-cell adherens junctions (AJs) consisting of at least nectin and l-afadin. Nectin is a Ca(2+)-independent homophilic immunoglobulin-like adhesion molecule, and l-afadin is an actin filament-binding protein that connects the cytoplasmic region of nectin to the actin cytoskeleton. Both the trans-interaction of nectin and the interaction of nectin with l-afadin are necessary for their colocalization with E-cadherin and catenins at AJs. Here, we examined the mechanism of interaction between these two cell-cell adhesion systems at AJs by the use of alpha-catenin-deficient F9 cell lines and cadherin-deficient L cell lines stably expressing their various components. We showed here that nectin and E-cadherin were colocalized through l-afadin and the COOH-terminal half of alpha-catenin at AJs. Nectin trans-interacted independently of E-cadherin, and the complex of E-cadherin and alpha- and beta-catenins was recruited to nectin-based cell-cell adhesion sites through l-afadin without the trans-interaction of E-cadherin. Our results indicate that nectin and cadherin interact through their cytoplasmic domain-associated proteins and suggest that these two cell-cell adhesion systems cooperatively organize cell-cell AJs.

Neurabin: a novel neural tissue-specific actin filament-binding protein involved in neurite formation.

We purified from rat brain a novel actin filament (F-actin)-binding protein of approximately 180 kD (p180), which was specifically expressed in neural tissue. We named p180 neurabin (neural tissue-specific F-actin- binding protein). We moreover cloned the cDNA of neurabin from a rat brain cDNA library and characterized native and recombinant proteins. Neurabin was a protein of 1,095 amino acids with a calculated molecular mass of 122,729. Neurabin had one F-actin-binding domain at the NH2-terminal region, one PSD-95, DlgA, ZO-1-like domain at the middle region, a domain known to interact with transmembrane proteins, and domains predicted to form coiled-coil structures at the COOH-terminal region. Neurabin bound along the sides of F-actin and showed F-actin-cross-linking activity. Immunofluorescence microscopic analysis revealed that neurabin was highly concentrated in the synapse of the developed neurons. Neurabin was also concentrated in the lamellipodia of the growth cone during the development of neurons. Moreover, a study on suppression of endogenous neurabin in primary cultured rat hippocampal neurons by treatment with an antisense oligonucleotide showed that neurabin was involved in the neurite formation. Neurabin is a candidate for key molecules in the synapse formation and function.

Ponsin/SH3P12: an l-afadin- and vinculin-binding protein localized at cell-cell and cell-matrix adherens junctions.

We recently isolated a novel actin filament (F-actin)-binding protein, afadin, that has two isoforms, l- and s-afadins. l-Afadin is ubiquitously expressed and specifically localized at zonula adherens (ZA) in epithelial cells and at cell-cell adherens junction (AJ) in nonepithelial cells, whereas s-afadin is abundantly expressed in neural tissue. l-Afadin has one PDZ domain, three proline-rich regions, and one F-actin-binding domain, whereas s-afadin lacks the third proline-rich region and the F-actin-binding domain. To understand the molecular mechanism of the specific localization of l-afadin at ZA in epithelial cells and at cell-cell AJ in nonepithelial cells, we attempted here to identify an l-afadin-binding protein(s) and isolated a protein, named ponsin. Ponsin had many splicing variants and the primary structures of two of them were determined. Both the two variants had three Src homology 3 (SH3) domains and turned out to be splicing variants of SH3P12. The third proline-rich region of l-afadin bound to the region of ponsin containing the second and third SH3 domains. Ponsin was ubiquitously expressed and localized at ZA in epithelial cells, at cell-cell AJ in nonepithelial cells, and at cell-matrix AJ in both types of cells. Ponsin furthermore directly bound vinculin, an F-actin-binding protein localized at ZA in epithelial cells, at cell-cell AJ in nonepithelial cells, and at cell-matrix AJ in both types of cells. Vinculin has one proline-rich region where two proline-rich sequences are located. The proline-rich region bound to the region of ponsin containing the first and second SH3 domains. l-Afadin and vinculin bound to ponsin in a competitive manner and these three proteins hardly formed a ternary complex. These results indicate that ponsin is an l-afadin- and vinculin-binding protein localized at ZA in epithelial cells, at cell-cell AJ in nonepithelial cells, and at cell-matrix AJ in both types of cells.

Afadin: A novel actin filament-binding protein with one PDZ domain localized at cadherin-based cell-to-cell adherens junction.

A novel actin filament (F-actin)-binding protein with a molecular mass of approximately 205 kD (p205), which was concentrated at cadherin-based cell-to-cell adherens junction (AJ), was isolated and characterized. p205 was purified from rat brain and its cDNA was cloned from a rat brain cDNA library. p205 was a protein of 1,829 amino acids (aa) with a calculated molecular mass of 207,667 kD. p205 had one F-actin-binding domain at 1,631-1,829 aa residues and one PDZ domain at 1,016- 1,100 aa residues, a domain known to interact with transmembrane proteins. p205 was copurified from rat brain with another protein with a molecular mass of 190 kD (p190). p190 was a protein of 1,663 aa with a calculated molecular mass of 188,971 kD. p190 was a splicing variant of p205 having one PDZ domain at 1,009-1,093 aa residues but lacking the F-actin-binding domain. Homology search analysis revealed that the aa sequence of p190 showed 90% identity over the entire sequence with the product of the AF-6 gene, which was found to be fused to the ALL-1 gene, known to be involved in acute leukemia. p190 is likely to be a rat counterpart of human AF-6 protein. p205 bound along the sides of F-actin but hardly showed the F-actin-cross-linking activity. Northern and Western blot analyses showed that p205 was ubiquitously expressed in all the rat tissues examined, whereas p190 was specifically expressed in brain. Immunofluorescence and immunoelectron microscopic studies revealed that p205 was concentrated at cadherin-based cell-to-cell AJ of various tissues. We named p205 l-afadin (a large splicing variant of AF-6 protein localized at adherens junction) and p190 s-afadin (a small splicing variant of l-afadin). These results suggest that l-afadin serves as a linker of the actin cytoskeleton to the plasma membrane at cell-to-cell AJ.

Nectin/PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein.

We have isolated a novel actin filament-binding protein, named afadin, localized at cadherin-based cell-cell adherens junctions (AJs) in various tissues and cell lines. Afadin has one PDZ domain, three proline-rich regions, and one actin filament-binding domain. We found here that afadin directly interacted with a family of the immunoglobulin superfamily, which was isolated originally as the poliovirus receptor-related protein (PRR) family consisting of PRR1 and -2, and has been identified recently to be the alphaherpes virus receptor. PRR has a COOH-terminal consensus motif to which the PDZ domain of afadin binds. PRR and afadin were colocalized at cadherin-based cell-cell AJs in various tissues and cell lines. In E-cadherin-expressing EL cells, PRR was recruited to cadherin-based cell-cell AJs through interaction with afadin. PRR showed Ca2+-independent cell-cell adhesion activity. These results indicate that PRR is a cell-cell adhesion molecule of the immunoglobulin superfamily which is recruited to cadherin-based cell-cell AJs through interaction with afadin. We rename PRR as nectin (taken from the Latin word "necto" meaning "to connect").

Afadin: A key molecule essential for structural organization of cell-cell junctions of polarized epithelia during embryogenesis.

Afadin is an actin filament-binding protein that binds to nectin, an immunoglobulin-like cell adhesion molecule, and is colocalized with nectin at cadherin-based cell-cell adherens junctions (AJs). To explore the function of afadin in cell-cell adhesion during embryogenesis, we generated afadin(-/-) mice and embryonic stem cells. In wild-type mice at embryonic days 6.5-8.5, afadin was highly expressed in the embryonic ectoderm and the mesoderm, but hardly detected in the extraembryonic regions such as the visceral endoderm. Afadin(-/-) mice showed developmental defects at stages during and after gastrulation, including disorganization of the ectoderm, impaired migration of the mesoderm, and loss of somites and other structures derived from both the ectoderm and the mesoderm. Cystic embryoid bodies derived from afadin(-/-) embryonic stem cells showed normal organization of the endoderm but disorganization of the ectoderm. Cell-cell AJs and tight junctions were improperly organized in the ectoderm of afadin(-/-) mice and embryoid bodies. These results indicate that afadin is highly expressed in the ectoderm- derived cells during embryogenesis and plays a key role in proper organization of AJs and tight junctions of the highly expressing cells, which is essential for proper tissue morphogenesis.

alpha-catenin-independent recruitment of ZO-1 to nectin-based cell-cell adhesion sites through afadin.

ZO-1 is an actin filament (F-actin)-binding protein that localizes to tight junctions and connects claudin to the actin cytoskeleton in epithelial cells. In nonepithelial cells that have no tight junctions, ZO-1 localizes to adherens junctions (AJs) and may connect cadherin to the actin cytoskeleton indirectly through beta- and alpha-catenins as one of many F-actin-binding proteins. Nectin is an immunoglobulin-like adhesion molecule that localizes to AJs and is associated with the actin cytoskeleton through afadin, an F-actin-binding protein. Ponsin is an afadin- and vinculin-binding protein that also localizes to AJs. The nectin-afadin complex has a potency to recruit the E-cadherin-beta-catenin complex through alpha-catenin in a manner independent of ponsin. By the use of cadherin-deficient L cell lines stably expressing various components of the cadherin-catenin and nectin-afadin systems, and alpha-catenin-deficient F9 cell lines, we examined here whether nectin recruits ZO-1 to nectin-based cell-cell adhesion sites. Nectin showed a potency to recruit not only alpha-catenin but also ZO-1 to nectin-based cell-cell adhesion sites. This recruitment of ZO-1 was dependent on afadin but independent of alpha-catenin and ponsin. These results indicate that ZO-1 localizes to cadherin-based AJs through interactions not only with alpha-catenin but also with the nectin-afadin system.

Different behavior of l-afadin and neurabin-II during the formation and destruction of cell-cell adherens junction.

We have recently isolated two novel actin filament-binding proteins, l-afadin and neurabin-II and shown that they are localized at cell-cell adherens junction (AJ) in epithelial cells. We found here that l-afadin, neurabin-II, ZO-1, and E-cadherin showed similar and different behavior during the formation and destruction of cell-cell AJ in MDCK cells. In MDCK cells, the accumulation of both l-afadin and E-cadherin, but not that of ZO-1, changed in parallel depending on Rac small G protein activity. Dissociation of MDCK cells by culturing the cells at 2 microM Ca2+ caused rapid endocytosis of E-cadherin, but not that of l-afadin or ZO-1. Addition of phorbol 12-myristate 13-acetate to these dissociated cells formed a tight junction-like structure where ZO-1 and l-afadin, but not neurabin-II or E-cadherin, accumulated. We furthermore found that, in non-epithelial EL cells, which expressed E-cadherin and attached to each other, l-afadin, neurabin-II, ZO-1 and E-cadherin were all localized at AJ. In cadherin-deficient L cells, I-afadin was mainly localized at cell-cell contact sites, but ZO-1 was mainly localized at the tip area of cell processes. Neurabin-II did not accumulate at the plasma membrane area. Neither l-afadin nor neurabin-II significantly interacted with alpha-, beta-catenin, E-cadherin, ZO-1 or occludin.

Two actions of frabin: direct activation of Cdc42 and indirect activation of Rac.

Frabin is an actin filament-binding protein which shows GDP/GTP exchange activity specific for Cdc42 small G protein and induces filopodium-like microspike formation and c-Jun N-terminal kinase (JNK) activation presumably through the activation of Cdc42. Frabin has one actin filament-binding (FAB) domain, one Dbl homology (DH) domain, first pleckstrin homology (PH) domain adjacent to the DH domain, one cysteine-rich FYVE domain, and second PH domain from the N-terminus to the C-terminus in this order. Different domains of frabin are involved in the microspike formation and the JNK activation, and the association of frabin with the actin cytoskeleton through the FAB domain is necessary for the microspike formation, but not for the JNK activation. We have found here that frabin induces the formation of not only filopodium-like microspikes but also lamellipodium-like structures in NIH3T3 and L fibroblasts. We have analysed the mechanism of frabin in these two actions and found that frabin induces filopodium-like microspike formation through the direct activation of Cdc42 and lamellipodium-like structure formation through the Cdc42-independent indirect activation of Rac small G protein. The FAB domain of frabin in addition to the DH domain and the first PH domain is necessary for the filopodium-like microspike formation, but not for the lamellipodium-like structure formation. The FYVE domain and the second PH domain in addition to the DH domain and the first PH domain are necessary for the lamellipodium-like structure formation. We show here these two actions of frabin in the regulation of cell morphology.

Extensive intestinal metaplasia in gastric carcinoma and in other lesions requiring surgery: a study of 3,421 gastrectomy specimens from dwellers of the Atlantic and Pacific basins.

BACKGROUND: Extensive intestinal metaplasia (EIM) has been reported in gastrectomies from patients dwelling in the Pacific and Atlantic basins. AIMS: To compare all the results in an attempt to explain the findings. METHOD: All sections from 3,421 gastrectomies were reviewed at various hospitals: 1946 in the Atlantic and 1475 in the Pacific basin. Sections with EIM showed IM encompassing one or more entire low power field (>or=5 mm in length/section) in one or more section. RESULTS: In the Atlantic basin, EIM was present in 18.8% (153 of 814) of specimens with intestinal carcinoma (IC) and in 10.3% (65 of 630) of those with diffuse carcinoma (DC). In the Pacific basin, EIM was found in 62.9% (412 of 655) of gastrectomies with IC and in 33.3% (160 of 481) of those with DC. The numbers of specimens with EIM were significantly higher in the Pacific than in the Atlantic basin for both carcinoma phenotypes, particularly among elderly patients (>or=60 years). CONCLUSIONS: The proportion of gastrectomies with EIM was higher among populations at a higher gastric cancer risk than in those with a lower cancer risk. EIM was mostly associated with IC rather than DC or with miscellaneous gastric diseases (841 control gastrectomies) in both basins. The proportion of gastrectomies with EIM was significantly higher in Vancouver than in New York and in Santiago de Chile than in Buenos Aires, even though these populations reside at approximately the same geographical latitude, but in different basins. Environmental factors seem to accelerate the evolution of EIM.

Gastric ciliated metaplasia. A study of 3406 gastrectomy specimens from dwellers of the Atlantic and the Pacific basins.

BACKGROUND: Ciliated cells in gastrectomies from patients dwelling in the Pacific and Atlantic basins have been reported previously. AIM: To compare all the results in an attempt to explain the findings. METHODS: Sections from 3406 gastrectomies were reviewed: 1966 and 1440 from the Atlantic and Pacific basins, respectively. Ciliated cells and intestinal metaplasia (IM) were recorded; IM was classified into focal or extensive IM. The total number of sections/gastrectomy was noted. RESULTS: In the Atlantic basin, 5% of specimens had ciliated metaplasia (CM); it was more frequent in intestinal carcinoma (IC; 9%) than diffuse carcinoma (DC; 3%) or miscellaneous gastric diseases (MGD; 3%). In the Pacific basin, the frequency of specimens with CM was 29%: it was more frequent in IC (43%) than in DC (16%) or MGD (10%). The difference between the frequency of CM in specimens with IC or with DC/MGD in the Atlantic and the Pacific basins was significant (p < or = 0.05). The presence of CM was influenced by age and the extent of IM in both basins, but not by sex or the number of sections investigated. CONCLUSIONS: CM-apparently an independent microscopic marker-was significantly higher in the Pacific than in the Atlantic basin. Environmental carcinogens involved in the evolution of IM and IC seem to be implicated in gastric ciliogenesis. Carcinogens that differ in nature and/or in strength in both basins might activate the latent natural genes encoding ciliated processes in gastric cells in patients subsequently developing gastric carcinoma, more notably of intestinal type.

Gastric glassy cells: a study of 3202 gastrectomy specimens from dwellers of the Atlantic and Pacific basins.

Fifteen years ago we detected gastric cells with glassy cytoplasm (GCs) in the human pyloric antrum. The frequency of these cells was subsequently investigated in sections from gastrectomies carried on in populations dwelling on the rim of the Atlantic and Pacific basins. In this work we compared the results obtained in these disparate geographic regions. We reviewed sections from 3203 gastrectomies (1942 in the Atlantic basin and 1261 in the Pacific basin). In the Atlantic basin 12/1942 (0.6%) of the gastrectomies had GCs, whereas in the Pacific basin 26/1261 (2.1%) of the gastrectomies had GCs. The difference was significant (p<0.05). The proportion of gastrectomies with GCs was higher in patients in Vancouver, Canada, than in New York, and higher in Santiago de Chile than in Buenos Aires, despite the fact that these populations reside at approximately the same geographic latitude. Previous studies with the same material indicated that both the extension of intestinal metaplasia and the frequency of ciliated metaplasia were significantly higher in the Pacific than in the Atlantic basin. Hence, the difference in the frequencies of GCs appears to be a new indication that dissimilar environmental exposures in the two basins might have influenced the histological make-up of the gastric mucosa.

Localization of l-afadin at puncta adhaerentia-like junctions between the mossy fiber terminals and the dendritic trunks of pyramidal cells in the adult mouse hippocampus.

We have recently found a novel cell-cell adhesion system at cadherin-based adherens junctions. This system consists of at least two components: nectin, an immunoglobulin-like cell adhesion molecule with Ca(2+)-independent homophilic binding activity, and l-afadin, an actin filament-binding protein that connects nectin to the actin cytoskeleton. In the present study, we investigated immunocytochemically the localization of l-afadin in the mouse hippocampus. At the light microscopic level, l-afadin immunoreactivity was demonstrated as flattened disks in the stratum lucidum of the CA3 area. By immunoelectron microscopy, signals for l-afadin were highly concentrated in a symmetrical manner at the puncta adhaerentia-like junctions between the mossy fiber terminals and the dendritic trunks of pyramidal cells. We furthermore immunostained the hippocampus with antibodies recognizing both l-afadin and s-afadin, a small splicing variant of l-afadin that is identical to AF-6. Immunoreactivity for l- and s-afadins was demonstrated not only as the flattened disks similar to that for l-afadin, but also as numerous fine dots widely distributed in all synaptic layers of the CA1 and CA3 areas. The latter finding may correspond with the recent report by Buchert et al. (1999, J. Cell. Biol. 144:361-371), who found that s-afadin (AF-6) and/or l-afadin was localized at the postsynaptic membranes of asymmetric synaptic junctions. Our present results indicate that l- and s-afadins are differentially distributed in the hippocampus and suggest that l-afadin localized at the puncta adhaerentia-like junctions in the mossy fiber terminals may regulate the structural and functional organization of these complex synaptic structures.

Total necrosis of hepatocellular carcinoma with a combination therapy of arterial infusion of chemotherapeutic lipiodol and transcatheter arterial embolization: report of 14 cases.

Combination therapy consisting of Lipiodol (Laboratoire Guerbet, Villepinte, France) containing styrene maleic acid neocarzinostatin and transcatheter arterial embolization (L-TAE) has been an important conservative therapy for hepatocellular carcinoma (HCC). We examined the clinical and pathologic characteristics of 14 HCC cases that achieved total tumor necrosis in response to L-TAE. The HCCs of all cases were resected 45 +/- 17 days after L-TAE and were confirmed to be totally necrotic. Ultrasonography showed a mean tumor size of 2.5 +/- 1.0 cm, often with a halo formation around the tumor. Angiographically, neovascularity and clear tumor stains were observed in all cases. Computed tomography portography showed nodular perfusion defects in all the cases examined. There were portal invasions in two cases. On Lipiodol-computed tomography, Lipiodol was densely and homogeneously retained within the whole tumor. The number of tumors was single in all diagnostic images. Macroscopic view of HCCs were single nodular type in nine cases and single nodular type with extra growth in four cases. Clear capsular formation was seen in each HCC nodule. Soft x-rays were taken to observe the exact distribution of Lipiodol in the operative specimens. Microscopic intrahepatic metastases were found histologically in four cases. Histologic examination showed the trabecular pattern with broad blood spaces in which Lipiodol was positive with Sudan III staining. Necrosis was seen not only in the main tumor, but also in the capsular invasions and microscopic metastases with Lipiodol deposition. The characteristics of the cases with total tumor necrosis were as follows. Deposition of Lipiodol throughout the tumor was essential, and clinically the cases showed a single HCC tumor with a diameter of more than 5 cm and arterial hypervascularity. The pathologic findings included expansive growth with capsular formation and trabecular-type HCC with abundant blood spaces. These findings are important for evaluating the radical efficacy of L-TAE.

Ischemic tolerance in hippocampal CA1 neurons studied using contralateral controls.

We induced ischemic tolerance unilaterally in gerbil hippocampus using the contralateral hippocampus as control. Ischemia for 2 min of right common carotid occlusion was reversible but sufficient to cause heat-shock protein 70 production in CA1 neurons. This pretreatment given four days prior to occlusion of both common carotids for 5 min, but not at longer preceding intervals, induced tolerance in right CA1 neurons. Neuroprotection was still evident two months after the 5 min occlusion. Adenosine triphosphate content and immunoreactive microtubule associate protein 2 in the hippocampus showed that the 5 min ischemic insult was essentially equal in both hemispheres. Repetitive pretreatments at two day intervals caused almost complete protection of CA1 neurons against subsequent 5 min ischemia, while a single pretreatment showed 80% protection. However, the increase in heat-shock protein 70 with repeated pretreatments was not significantly more than with one pretreatment. We concluded that true ischemic tolerance was induced by ischemic stress itself, was long-lasting, was not due to mitigation of subsequent ischemia, and was augmented by repetition without further increase of heat-shock protein 70.

Ischemic damage and subsequent proliferation of oligodendrocytes in focal cerebral ischemia.

In order to achieve a better understanding of the pathophysiology of ischemic white matter lesions, oligodendrocytic degeneration and subsequent proliferation were examined in the mouse model of middle cerebral artery occlusion. In situ hybridization histochemistry for proteolipid protein messenger RNA was employed as a sensitive and specific marker of oligodendrocytes, and immunohistochemistry for myelin basic protein was used as a compact myelin marker. Immunohistochemistry for microtubule-associated protein 2 and albumin was employed to monitor neuronal degeneration and the breakdown of the blood brain barrier, respectively. In the ischemic core of the caudoputamen, the immunoreactivity for microtubule-associated protein 2 disappeared and massive albumin extravasation occurred several hours after vessel occlusion, while proteolipid protein messenger RNA signals remained relatively strong at this time. The messenger RNA signals began to attenuate 12 h after ischemia and were hardly detectable 24 h after ischemia in the whole ischemic lesion. In situ end-labeling of fragmented DNA showed some cells with proteolipid protein messenger RNAs to have DNA fragmentation at this period. In contrast to proteolipid protein messenger RNA signals, the immunoreactivity for myelin basic protein was detected as long as five days after ischemia. An apparent increase in the cells possessing strong proteolipid protein messenger RNA signals was found five days after ischemia, mainly in the corpus callosum and the cortex bordering the infarcted areas. A double simultaneous procedure with in situ hybridization for proteolipid protein messenger RNA and immunohistochemistry for glial fibrillary acid protein or lectin histochemistry for macrophages/microglia showed proliferating oligodendrocytes to be co-localized with reactive astrocytes and macrophages/microglia. These findings show that oligodendrocytic damage occurred following ischemic neuronal damage and the breakdown of the blood brain barrier, but preceded the breakdown of myelin proteins in the ischemic lesion, that an apoptosis-like process was involved in ischemic oligodendrocytic death, and that surviving oligodendrocytes responded and proliferated in the outer border of the infarcted area.

Expression of cripto-1 in human colorectal adenomas and carcinomas is related to the degree of dysplasia.

The expression and localization of cripto-1 (CR-1) and epidermal growth factor receptor (EGFR) were assessed by immunocytochemistry in 41 human colorectal carcinomas, 57 adenomas, 9 hyperplastic polyps and in 98 noninvolved colonic mucosa samples that were adjacent to adenoma and/or carcinoma. Thirty-two (78.0%) and 19 (46%) carcinomas showed staining for CR-1 and EGFR, respectively, whereas 24 (42.0%) and 25 (43.8%) of the adenoma samples were reactive with the anti-CR-1 and anti-EGER antibodies, respectively. Two (22.2%) and 1 (11.1%) of the hyperplastic polyps demonstrated moderate levels of staining with anti-CR-1 and anti-EGFR antibodies. In contrast, none of the normal, noninvolved colonic mucosa samples reacted with the CR-1 antibody, whereas only 1 (1.0%) reacted with the EGFR antibody. Between EGFR and CR-1 expression, there was no significant association within either adenomas or carcinomas. A significant difference in the incidence for CR-1 expression was observed between adenomas and carcinomas (p<0.001). Within adenomas, the frequency of CR-1 was related to the histological degree of atypia. Immunostaining for p53 was also observed in 10 (24%) of the carcinomas, in 10 (17%) of the adenomas and in none of the hyperplastic polyps nor colonic mucosa samples. No statistically significant difference for p53 staining was observed between the adenomas and carcinomas. However, adenomas with moderate atypia exhibited relatively strong positive staining for p53 (p<0.05) compared to either adenomas with mild or severe atypia. A slight trend (p<0.05) for coexpression of p53 and CR-1 was detected in adenomas but not in carcinomas. These data demonstrate that CR-1 is a tumor marker for colon carcinomas and additionally that the expression of CR-1 may be an important factor in the early stages of colon cancer development during the adenomacarcinoma transition.

Immunohistochemical detection of ribonucleotide reductase in human breast-tumors.

Ribonucleotide reductase (RNR) consists of two non-identical subunits, R1 and R2 and is one of the key enzymes involved in DNA biosynthesis. RNR activity is considerably higher in malignant tumors than in normal tissues in the rat suggesting that RNR may play an important role in the pathogenesis of human tumors. In order to obtain immunological reagents to study the localization and level of expression of RNR in various human tissues, a synthetic peptide containing sequences corresponding to the COOH-terminal region of the human R2 subunit was used to generate rat monoclonal antibodies. The generated rat monoclonal antibodies (IgG) inhibited RNR enzymatic activity purified from murine P388 leukemia cells. These antibodies were used to immunohistochemically examine the distribution of RNR in a small panel of 8 malignant and 4 benign human breast tumors. Positive immunostaining for RNR was observed in the cytoplasm of human breast carcinoma cells in which a specific 44 kDa specific band of R2 subunit was also detected by Western blot analysis. The immunostaining was blocked by preabsorption of the antibody with an excess amount of the synthetic peptide immunogen. In 8 of 8 breast carcinomas, positive immunostaining for the R2 subunit was observed whereas noninvolved, adjacent breast tissue showed no staining with this antibody. In addition, few of the benign breast lesions exhibited staining with this antibody. These data indicate that these antibodies can immunohistochemically detect RNR in frozen or formalin-fixed, paraffin- embedded tissues and that there is a differential expression of RNR between breast tumors and non-involved breast tissue. Immunohistochemical detection of RNR using these antibodies may therefore be useful for the diagnosis of human breast tumors.

Immunohistochemical detection of cripto-1, amphiregulin and transforming growth-factor-alpha in human gastric carcinomas and intestinal metaplasias.

The expression and localization of cripto-1 (CR-1), amphiregulin (AR) and transforming growth factor alpha (TGFalpha) were assessed by immunocytochemistry in 37 primary human gastric tumors, 30 noninvolved gastric mucosa samples that were adjacent to carcinoma but exhibited intestinal metaplasia and 37 adjacent, noninvolved gastric mucosa samples. Seventeen (46%), nineteen (51%) and twenty-one (57%) carcinomas showed staining for CR-1, AR and TGFalpha, respectively; whereas sixteen (53%), eight (26%) and five (17%) intestinal metaplasias were reactive with the anti-CR-1, anti-AR and anti-TGFalpha antibodies, respectively. In contrast, none of the normal, noninvolved gastric mucosa samples reacted with the TGFalpha antibody and only 1 (3%) of these samples showed weak staining with the anti-CR-1 antibody. However, 8 (21%) of the normal gastric mucosa samples showed moderate levels of staining with the AR antibody. Within the carcinomas, there was a slight trend for association between TGFalpha and CR-1 expression (p<0.05), but no correlation was found between epidermal growth factor receptor and CR-1 expression. Staining for p53 was observed in 26 (70%) of the carcinomas, 3 (10%) intestinal metaplasias and none of the gastric mucosa samples. This data demonstrate that CR-1, like TGFalpha, may be a tumor marker for a subset of gastric carcinomas in addition to being an important factor in the early stages of gastric cancer development.