Peter Herrlich

CD44 variant exon epitopes in primary breast cancer and length of survival

CD44 designates a group of closely related cell-surface proteins generated by alternative splicing. We have previously shown that splice variants carrying sequences encoded by exon v6 are preferentially expressed in metastatic animal cancer cell lines and that they confer metastatic behaviour on non-metastatic animal tumour cell lines. In this study we set out to assess the expression of CD44 epitopes specific for variant exon sequences in human breast cancer and their potential for determining prognosis. We used affinity-purified polyclonal sera and four monoclonal antibodies raised against the human homologues of CD44 variant exon sequences to investigate the presence of CD44 on 100 primary invasive breast tumours, 12 local recurrences, 18 lymph node metastases, and normal tissue controls. Whereas normal mammary ductal epithelial cells and cells derived from hyperplastic lesions do not express CD44 variant exons, expression of v3, v5, and v6 epitopes was found in most tumour samples. The DIII (exon v6) epitope was present in 84% of the primary tumours and in 100% of axillary lymph node metastases and local recurrences. The presence of these CD44 epitopes is correlated with poor overall survival. 15 patients with exon-v6-negative tumours had good survival compared with 76 patients with exon-v6-positive tumours (p = 0.005; log rank test). Multivariate analysis showed that the CD44 epitope encoded by exon v6 was a good marker for prognosis independent of progesterone receptor, lymph node status, tumour size, and grade.

Heterodimer formation of cJun and ATF-2 is responsible for induction of c-jun by the 243 amino acid adenovirus E1A protein

The adenovirus E1A proteins differentially regulate AP‐1‐responsive genes. Collagenase and stromelysin are repressed by E1A, whereas the expression of c‐jun is elevated. Inhibition of collagenase has been found to be exerted through the consensus AP‐1 binding site TGAGTCA. Here we show that the distal AP‐1 binding site in the c‐jun promoter, the jun2TRE (TTACCTCA), is the decisive element of this promoter in mediating the positive response to the 243 amino acid E1A product. In vitro binding studies revealed that, in contrast to the consensus AP‐1 site which is preferentially targeted by dimers composed of the Jun and Fos families, the jun2TRE binds heterodimers composed of cJun and ATF‐2(‐like) proteins. Since stimulation of c‐jun transcription is a function of the transforming domain of E1A encoded by conserved region 1, cJun‐‐ATF‐2 may be one of the effector factors involved in transformation. The data further suggest that E1A can distinguish between cJun‐‐cJun and cJun‐‐ATF‐2 in imposing opposite states of activity.

The fos and jun families of transcription factors

The AP-1 family plays crucial roles in cell growth, proliferation, differentiation and apoptosis. It is the endpoint of several pathways of signal transduction, including one which triggers cancerous growth. The control of its activity is an issue both of basic science and of therapy for cancer and other diseases. Chapters are written by European experts in different areas. AP-1 is looked at as a factor regulating genes involved in metastatic properties of cancer, as a factor addressed by viral gene products and by steroid and retinoic acid receptors during the process of anti-inflammation. It covers the important role of AP-1 in cell transformation, induced by either chemical or physical mutagens or by expression of oncogenes.

Control of gene expression in bacteriophage T7: Transcriptional controls

SummaryTwo transcriptional control mechanisms of T7 can be distinguished both affecting the transcription by E. coli RNA polymerase: An early control and an “early-late” control. In wild type infections, both transcriptional control proteins appear at approximately the same time. Mutations in the early control gene have, therefore, little effect on transcription, if tested in the presence of virus RNA polymerase. Using mutants in T7 RNA polymerase, the appearance of the “early-late” control is delayed. Then, the effect of the early control gene is dramatic, its deficiency leading to an overproduction of host and early T7 RNA. The early RNA control appears to be exerted by the T7 protein kinase, the “early-late” control protein is most likely identical with the transcriptional inhibitor, which has been isolated and purified (Ponta et al., 1974). Both control proteins inhibit the initiation of RNA synthesis by E. coli RNA polymerase.

E. coli membranes become permeable to ions following T7-virus-infection.

SummaryInfection of E. coli with the viruses T7 or T3 leads to a dramatic efflux of potassium ions. This ion efflux is caused by the virus particle since no concomitant protein synthesis is required. T7 mutants carrying deletions in the M-gene (Schweiger et al., 1975), however, yield virus particles disturbed in the ion release.

Ribosomes after infection with bacteriophage T4 and T7.

SummaryThe synthesis of E. coli ribosomal proteins ceases after infection with bacteriophages T4 or T7 as does the synthesis of most other host proteins. The shut-off does not affect all ribosomal proteins to the same extent. After T7 infection no new proteins were detected in NH4Cl-washed ribosomal particles. Bacteriophage T4, however, induces 3–4 new protein bands demonstrated by one-dimensional gel electrophoresis. The appearance of these bands is prevented by the addition of rifampicin at the time of infection but not when rifampicin is added one minute after infection. The NH4Cl-washed ribosomal particles present at the time of T7 or T4 infection do not show any structural changes by sedimentation, subunit dissociation, or protein analysis on two-dimensional polyacrylamide gels. However, by labeling the T7 infected cells with 32P-phosphate, it is seen that the ribosomes become phosphorylated. The 32P-label comigrates with ribosomal proteins. This phosphorylating activity depends on a T7 gene. The T7 protein phosphokinase utilizes ribosomes as phosphate acceptor in vitro. The T7 ribosomes (NH4Cl-washed) still function in vitro as do ribosomal particles from uninfected cells.

Cd44 and Splice Variants of Cd44 in Normal Differentiation and Tumor Progression

CD44, originally defined by antibodies as a leukocyte surface protein (reviewed in Haynes et al., 1989), has become a polymorphic family of proteins expressed in various cells and conditions. The polymorphism is due to differential modifications and to splice variation (Hughes et al., 1983; Omary et al., 1988; Kansas et al., 1989; Picker et al., 1989; Goldstein et al., 1989; Goldstein and Butcher, 1990; Stamenkovic et al., 1991, Dougherty et al., 1991; Brown et al., 1991; Gunthert et al., 1991; Shtivelman and Bishop, 1991; Jackson et al., 1992). Because of the polymorphism a multitude of functions can be expected. Yet these need t be defined. All or part of the CD44 glycoproteins have affinity for hyaluronic acid, some forms are linked to chondoitin sulfate and bind fibronectin and collagen (Aruffo et al., 1990; Miyake et al., 1990; Stamenkovic et al., 1989; Goldstein et al., 1989; Wolffe et al. 1990; Carter and Wayner, 1988; Jalkanen and Jalkanen, 1992). These affinnites may have functions in association with different primary structures domains introduced by splice variation.

An Update of the Mammalian UV Response: Gene Regulation and Induction of a Protective Function

Bacteria and yeast react to DNA damaging agents by synthesizing more of several gene products (Walker, 1985; Moustacchi, 1987; Friedberg, et al., 1988). Among the induced products there are DNA repair functions and proteins required for mutagenesis. For mammalian cells, formally a similar response has been detected. New gene products are synthesized (Herrlich, et al., 1986). No DNA repair induction has yet been characterized although it is likely that it exists since viral and shuttle vector probes have revealed enhanced reactivation and mutagenesis (Radman, 1980; Cornelis, et al., 1982; Defais, et al., 1983; Sarasin, 1985; Dion and Hamelin, 1987; Herrlich, 1988a; Protic, et al., 1988). Also, low doses of mutagens may, under certain conditions, modify subsequent mutagenic and clastogenic responses (Samson and Schwartz, 1980; Kaina, 1982, 1983; Rieger, et al., 1982; Olivieri, et al., 1984; Laval and Laval, 1984). Fluctuation analyses supplied support for the idea that proneness to mutation is passed on to daughter cells (Maher, et al., 1988). As another parallel between bacteria and mammalian cells DNA damage causes reinitiation of replication (Kogoma, et al., 1979) which in mammalian cells is observed as gene amplification (Lavi, 1981; Brown, et al., 1983; Schimke, 1984) or recruitment of quiescent cells into the cell cycle (Cohn, et al., 1984). Molecular genetic techniques have made both the mechanisms of signal transduction and gene functions experimentally accessible. Here, current ideas on damage-induced gene expression and its phenotypic consequences are presented.

Early T7 gene expression: rates of RNA synthesis and degradation, protein kinase dependent termination of transcription, and efficiency of translation.

SummaryThe mode of transcription of early T7 genes starting from one promotor region and generating a unique polycistronic RNA species suggests the appearance of equimolar amounts of the monocistronic species after RNA processing. Rate measurements revealed, however, a disproportion in the generation of the individual early RNA species. The rate of appearance of the promotor-proximal M gene message (nomenclature see in Table 1) is 4–5x the rate of appearance of all other species. This rate pattern is caused by termination behind the M gene because i) the rate of RNA degradation is fairly similar for most RNA species and ii) termination behind the M gene is released in a T7 mutant lacking protein kinase or in wild type infections in the absence of protein synthesis. Then, the RNA species are produced in equimolar amounts.The rate of degradation is similar for all early RNA species except for the protein kinase message. As measured by two independent methods, the physical halflives of M, POL, 1.1, and LIG (nomenclature see Table 1) message were 7–8 min (30°), while KIN RNA was degraded with a halflife of 4 min. The functional halflives were around 50% of the physical halflives. There is apparently no relationship between size of RNA and halflife, and the data suggest specific signals on each RNA which determine the rate of degradation.The monocistronic RNA species are utilized with different rates in translation. The M gene is not only transcribed more often, it is also translated with highest efficiency. The in vivo translation of the POL gene message occurred with the lowest rate.

Host- and phage-RNA polymerase mediated synthesis of T7 lysozyme in vivo

SummaryThe lysozyme gene T7 can be transcribed in vivo by E. coli polymerase and by T7 phage polymerase, classifying this gene as “early/late”.