Involvement of c-JUN in the regulation of terminal differentiation genes in normal and malignant keratinocytes.

In stratifying cultures of human keratinocytes, expression of the proto-oncoprotein c-JUN and the small proline rich 2 (SPRR2) protein, a precursor of the cornified cell envelope, are inversely related. Whereas c-JUN is typically found in basal proliferating cells, SPRR2 is restricted to suprabasal differentiating layers. Malignant keratinocytes (derived from squamous cell carcinoma, SCC) have reduced sprr2 expression, consistent with their low potential to differentiate, and express c-jun at higher levels than normal keratinocytes. A direct relation between c-jun and sprr2 expression was shown in several ways: transient ectopic expression of c-jun inhibits sprr2a promoter activity in normal differentiating cells, whereas in malignant keratinocytes a dominant negative c-jun mutant restored at least partially both the low promoter activity and the expression of endogenous sprr2. These effects are mediated via a 134 bp promoter fragment which does not include the sprr2a AP-1 binding site. Interestingly, in an SCC cell line, constitutively expressing the dominant c-jun mutant, expression of the terminal differentiation marker involucrin is also strongly increased, suggesting that c-JUN is a general modulator of keratinocyte terminal differentiation rather than only affecting the expression of sprr2.

Expression of the SPRR cornification genes is differentially affected by carcinogenic transformation.

The small proline rich protein (SPRR) genes constitute a family of conserved genes which are part of the human epidermal differentiation complex (EDC) on chromosome 1q21 and code for precursor proteins of the cornified cell envelope. The expression of these genes is strictly linked to keratinocyte terminal differentiation both in vivo and in vitro. Here we show that cultured cell lines derived from squamous cell carcinoma (SCC) show significantly lower levels of SPRR expression than normal human keratinocytes. However, the residual SPRR expression in SCC lines appears to be both gene and cell line specific. Expression of SPRR2 appears to correlate well with the residual ability of these cells to differentiate. However, the kinetics of SPRR2 expression, following treatment with calcium, an inducer of keratinocyte differentiation, are typical for each cell line and differ substantially from the ones found in normal cells. In most cell lines a rapid transient expression of SPRR2 contrasts with a slow induction leading to a high sustained level of expression in normal cells. This pattern of expression is typical for SPRR2 and not observed for the other SPRR genes or involucrin. Our analysis indicates that the expression of various keratinocyte terminal differentiation markers, even when involved in the same biological process (cornification), can be differentially affected by carcinogenic transformation.

Nucleotide sequence of the tryptophan decarboxylase gene of Catharanthus roseus and expression of tdc-gusA gene fusions in Nicotiana tabacum.

The enzyme tryptophan decarboxylase (TDC; EC 4.1.1.28) converts tryptophan into tryptamine. In Catharanthus roseus and other plants capable of producing terpenoid indole alkaloids (TIAs) TDC links primary metabolism to the secondary metabolic pathway involved in the biosynthesis of these compounds. The accumulation of tdc mRNA in C. roseus cells is developmentally regulated and transcriptionally influenced by elicitors (induction) and auxins (repression). Here we report that TDC is encoded by a single copy gene in the C. roseus genome. No introns were observed upon isolation and sequencing of this gene. To study gene expression controlled by the tdc promoter, a 2 kb promoter fragment and a number of 5' deleted promoter derivatives were joined in translational fusion to a beta-D-glucuronidase reporter gene (gusA). Expression of the chimaeric constructs was monitored in stably transformed tobacco plants and in transiently transfected tobacco protoplasts. Histochemical and fluorimetric analysis of transgenic plants revealed that 1938 bp of the tdc promoter (with respect to the translational start codon) give rise to GUS activity in roots, stems and leaves. No tissue or cell type specificity was noted. Promoter deletions up to nucleotide -398 directed lower levels of gusA expression but conferred the same pattern of staining for GUS activity as the -1938 construct. Further deletion of the tdc promoter up to nucleotide -232 resulted in drastically reduced GUS activity levels and loss of GUS staining in all parts of the transgenic plants. In contrast to stable transformation, the -232 tdc-gusA construct gave rise to GUS activity levels comparable to those of the -398 construct in an assay system for transient expression in protoplasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Physical and genetic mapping of a novel chromosome 19 ERCC1 marker showing close linkage with myotonic dystrophy.

Recent genetic linkage analyses have mapped the myotonic dystrophy locus to the region of 19q13.2-13.3 lying distal to the gene for creatine kinase subunit M (CKM). The human excision repair gene ERCC1 has also been mapped to this region of chromosome 19. A novel polymorphic DNA marker, pEO.8, has been isolated from a chromosome 19 ERCC1-containing cosmid that maps to a 300-kb NotI fragment encompassing both CKM and ERCC1. Genetic linkage analysis reveals close linkage between pEO.8 and myotonic dystrophy (DM) (zmax = 19.3, theta max = 0.01). Analysis of two key recombinant events suggests a mapping of DM distal to pEO.8 and CKM.