BRK/Sik expression in the gastrointestinal tract and in colon tumors.

Clones encoding the breast tumor kinase BRK were isolated from a normal human small intestinal cDNA library that was screened with the cDNA encoding the mouse epithelial-specific tyrosine kinase Sik. Although BRK and Sik share only 80% amino acid sequence identity, Southern blot hybridizations confirmed that the two proteins are orthologues. Sik was mapped to mouse distal chromosome 2, which shows conservation of synteny with human chromosome 20q13.3, the location of the BRK gene. BRK expression was examined in the normal gastrointestinal tract, colon tumor cell lines, and primary colon tumor samples. Like Sik, BRK is expressed in normal epithelial cells of the gastrointestinal tract that are undergoing terminal differentiation. BRK expression also increased during differentiation of the Caco-2 colon adenocarcinoma cell line. Modest increases in BRK expression were detected in primary colon tumors by RNase protection, in situ hybridization, and immunohistochemical assays. The BRK tyrosine kinase appears to play a role in signal transduction in the normal gastrointestinal tract, and its overexpression may be linked to the development of a variety of epithelial tumors.

Ryk is expressed in a differentiation-specific manner in epithelial tissues and is strongly induced in decidualizing uterine stroma.

Ryk is a ubiquitously expressed tyrosine kinase-like receptor of unknown activity and associations. We examined ryk expression in adult mouse epithelial tissues and during embryonic development at the histological level. Ryk RNA is present at greatly increased levels in cells at particular stages of epithelial differentiation: the basal layer of skin and tongue epithelia, the intervillous layer and some crypt bases of the intestine and the lower matrix region of the hair follicle. Although ryk RNA is expressed at similar levels in a variety of tissues from embryonic day 10.5 to 18.5, specific induction of ryk RNA can be seen by in situ hybridization in the basal layer of skin and hair follicle at day 15.5-16.5, and protein staining localizes to the hair follicle by immunohistochemistry. At day 4.5 and 6.5, little if any ryk is present in the blastocyst, but it is transiently induced at a high level in mature decidual cells of the uterine stroma. We review a number of independent isolations of ryk, including fruit fly and nematode members of the ryk family. Because ryk is induced in epithelial cells seeking a final place in a differentiated tissue, or during remodeling of the endometrium, and a homologous gene, derailed, is known to regulate muscle and nerve target seeking in Drosophila, ryk may also be involved in cellular recognition of appropriate context.

p53-independent induction of p21WAF1/CIP1 expression in pericentral hepatocytes following carbon tetrachloride intoxication.

The cyclin-dependent kinase, proliferating cell nuclear antigen, and stress-activated protein kinase/c-jun NH2 terminal kinase inhibitor p21WAF1/CIP1 can induce G1 arrest, and its expression coincides with the cessation of replication in many systems. We examined expression of p21 during the early stages of carbon tetrachloride intoxication in the mouse liver and observed a dramatic increase in p21 RNA levels between 4 and 8 h after administration. p21 expression, visualized by in situ hybridization, is induced in pericentral hepatocytes before carbon tetrachloride-induced necrosis. Examination of c-fos and c-myc expression patterns confirm that these immediate-early genes are induced in similar regions of the mouse liver. p21 induction is not dependent on p53; we observed similar levels and localization of p21 in wild-type and p53 null animals. Immunohistochemical localization of p21 and CCAAT/enhancer-binding protein expression shows that p21 protein accumulation is limited to a subset of CCAAT/enhancer-binding protein-positive hepatocytes. A second peak of periportal and intermediate zone-specific p21 gene expression, appearing 1-2 days after injection, is also p53 independent and may represent cell cycle checkpoints or postmitotic growth arrest. Sporadic p21 expression was also detected in pairs of hepatocytes distributed throughout the liver acini in healthy animals. Together, these data suggest several roles for p21 in the liver in response to toxicity, regeneration, and growth inhibition.

p21--negative regulator of the cell cycle.

Progression through the cell cycle is regulated by cyclins and cyclin-dependent kinases (Cdks). The cyclin kinase inhibitor p21 (also known as WAF1, CIP1, SDI1, and MDA-6) can induce G1 arrest and block entry into S phase by inactivating Cdks or by inhibiting activity of proliferating cell nuclear antigen (PCNA). In normal cells, p21 exists in quaternary complexes with cyclin, Cdk, and PCNA. Transcription of the p21 gene is activated by p53-dependent and -independent mechanisms. Mice deficient in p21 exhibit no apparent phenotype, although p21 function has been demonstrated to be necessary for p53-mediated G1 arrest following irradiation of p21-deficient mouse embryonic fibroblasts. Thus, the function of p21 under normal circumstances appears to be redundant. p21 is expressed in terminally differentiating cells of a variety of tissues in a p53-independent manner. Overexpression of p21 results in G1 arrest and has been shown to suppress effectively tumor growth in vitro and in vivo. We review the recent literature describing the functional characterization of p21. This protein plays a key role in regulating the cell cycle and may have potential gene therapy applications.

p21 (WAF1/CIP1) expression is induced in newly nondividing cells in diverse epithelia and during differentiation of the Caco-2 intestinal cell line.

We examined the relationship between expression of the p21 (WAF1/CIP1) inhibitor of cyclin-dependent kinases, cessation of proliferation, and terminal differentiation in the epithelia of the gastrointestinal tract. Using in situ hybridization, we performed a detailed study of patterns of p21 mRNA expression in different regions of the stomach, along the length of the intestine, and in tongue, cervix, and hair follicle. We detected strong hybridization only in cells that had ceased proliferation and begun the process of terminal differentiation. Induction of p21 transcription may serve as a useful marker for dissection of differentiation programs in these diverse epithelia. To determine the relative levels of p21 expressed in various regions of the gastrointestinal tract from the esophagus to the colon, we used quantitative RT-PCR with endogenous and exogenous sequences as internal standards. The highest levels of p21 expression were detected in the distal small intestine. To further investigate the role that cell cycle regulation may play during differentiation of intestinal epithelial cells, we examined the expression of p53, p21, cyclin D1, cyclin E, and E2F1 in the Caco-2 colon carcinoma cell line, which differentiates spontaneously after reaching confluence. p21 and p53 mRNA and protein levels increase as Caco-2 cells differentiate. In both undifferentiated and differentiated Caco-2 cells, p53 protein was not inducible by DNA damaging agents, suggesting the absence of functionally wildtype protein. Caco-2 cells should provide a useful model system for studying regulation of p21 and determining if it plays a role during intestinal epithelial cell differentiation.

A novel intracellular epithelial cell tyrosine kinase is expressed in the skin and gastrointestinal tract.

A portion of the catalytic domain of a novel tyrosine kinase was cloned from mouse intestinal crypt cells, in a screen designed to identify kinases that may play a role in the regeneration of the intestinal epithelium (E Siyanova, MS Serfas, IA Mazo and AL Tyner, Oncogene 9, 2053-2057). We have cloned a cDNA encoding this kinase, termed sik for src-related intestinal kinase. The sik cDNA encodes a 451 amino acid protein that shares 80% identity with the recently cloned human tyrosine kinase, brk. Sequences found in src family kinases, such as SH2 and SH3 domains and a putative regulatory tyrosine at the carboxy terminus are found in the sik kinase. In contrast, sik lacks a myristylation site. The protein encoded by the sik cDNA has tyrosine kinase activity when expressed in E. coli. We have determined that sik is expressed only in epithelial tissues, including the skin and lining of the alimentary canal, and using in situ hybridization we show that expression of sik mRNA is restricted to the cell layers immediately above the proliferative cell zone in these epithelia. The sik mRNA is first detected at day 15.5 of gestation in the mouse embryo, where it is expressed in the newly forming granular layer of the skin. The restricted expression of sik to differentiating cells of rapidly renewing epithelia suggests that sik may play a specialized role in these tissues.

Tyrosine kinase gene expression in the mouse small intestine.

To identify tyrosine kinases that may regulate regeneration of the mammalian intestinal epithelium, we amplified portions of the catalytic domains of protein kinases expressed in intestinal crypt cells, using the polymerase chain reaction technique with primers directed against two invariant amino acid sequence motifs found in all kinases. These fragments were cloned and a library of kinase catalytic domains was generated. Sequence analysis of unique clones resulted in the identification of the catalytic domains of several characterized tyrosine kinases, including lyn, hck, c-fgr, tec, JAK2, itk, and the putative receptor kinase ryk, and expression of these kinases has not previously been described in the intestine. We compared the levels of mRNA encoding these kinases in multiple tissues using RNase protection assays, and we localized the expression of hck, lyn, and JAK2 in the intestine using in situ hybridization. In addition, we identified two novel putative catalytic domain sequences. One of these, which we have named sik (src-related intestinal kinase), is expressed at high levels in the gastrointestinal tract and may play a specific role in signal transduction in epithelial tissues.

HNF-1 alpha and HNF-1 beta expression in mouse intestinal crypts.

Hepatocyte nuclear factors 1 alpha and 1 beta (HNF-1 alpha and HNF-1 beta) are homologous homeodomain-containing transcription factors that form homodimers or heterodimers that bind the same consensus sequence in the promoters of several genes expressed in liver. Many of these genes, including the mouse alpha-fetoprotein (AFP) gene, are also expressed in the intestinal epithelium. AFP expression is restricted to the enteroendocrine cell lineage in the adult small intestine, and we have investigated the distribution of HNF-1 alpha and HNF-1 beta mRNA in the mouse intestine to determine whether expression of either of these regulatory factors colocalizes with AFP. We found that transcripts encoding both factors are expressed at the highest levels in crypts of small and large intestine. Quantitation shows that the relative ratio of HNF-1 alpha to HNF-1 beta mRNAs appears lower in the colon, where AFP is not expressed. Alterations in the ratio of HNF-1 alpha to HNF-1 beta along the length of the intestine may influence HNF-1 dimer formation and expression of target genes. Although HNF-1 is necessary for transcription of the AFP gene, other factors must be involved in eliciting the cell type-specific pattern of AFP expression in the intestine.