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Abundance and activity of p53 are predominantly regulated posttranslationally. Structural disturbance in transcribed genes induced by radiation, e.g. DNA damage, or by transcriptional inhibitors cause p53 protein stabilization by a yet unknown mechanism. Using stable and transient transfections for the analysis of p53 mutant proteins, we have ruled out a role in stabilization by UV, gamma irradiation or actinomycin C for the following putative phosphorylation sites in the p53 protein: serines 6, 9, 15, 33, 315 and 392, and threonine 18. By double mutation combinations of phosphorylations were also ruled out; 6,9; 15,18; 15,37. These mutations eliminate modifications by casein kinases I and II, DNA-PK, ATM, CDK and JNK. Also the 30 carboxyterminal amino acids are not required for induced p53 stabilization. Thus neither phosphorylations of individual amino acids nor interactions of the carboxyterminus of p53 with cellular macromolecules appear to play a role in the stabilization process. The only single prerequisite for induced stabilization of p53 is its prior destabilization by Mdm2. However, the level of active Mdm2 must be controlled carefully: overexpression of Mdm2 inhibits UV induced p53 stabilization.

Mammalian cells in culture react to ultraviolet irradiation with the massive transcriptional activation of several genes and with the stabilization of the p53 protein. While U.V.-induced transcription of several immediate-response genes depends on U.V.-induced activation of signal transduction generated by non-nuclear mechanisms, stabilization of p53 and the transcription of several delayed-response genes are triggered by U.V.-induced DNA damage. By comparing dose responses for the activation by U.V. of delayed-responsive genes (collagenase 1, metallothionein IIA) in cells from patients with different DNA repair deficiencies (complementation groups of Xeroderma pigmentosum, Cockayne's syndrome and Trichothiodystrophy), we show here that U.V.-induced transcription of these genes does depend on pyrimidine dimers in transcribed regions of the genome (but not on damage in its silent part). Since all cells with defects in DNA repair that had been tested and which lack different enzymes, respond to U.V. with expression of these same genes, functional repair does not appear to be required for the induction of expression, and repair intermediates (which would not be identical in cells of different repair deficiency) cannot be responsible for signal generation.

The neurofibromatosis 2 (NF2) tumor suppressor gene encodes an intracellular membrane-associated protein, called merlin (or schwannomin), that belongs to the band 4.1 family of cytoskeleton-associated proteins. Inactivating NF2 mutations occur in several sporadic tumor types and have been linked to the NF2 disease, whose hallmark is the development of bilateral Schwann cell tumors (schwannomas) of the eighth cranial nerve. Two major alternatively spliced NF2 variants are expressed in normal tissues: 'NF2-17' lacking exon 16 and 'NF2-16' that contains exon 16 and encodes a merlin protein truncated at the C-terminus. We report that overexpression of NF2-17 in rat schwannoma cells inhibits their growth in vitro and in vivo, while NF2-16 fails to influence schwannoma growth. Tumor growth inhibition by merlin depends on an interdomain association occurring either in cis or in trans between the N- and C-termini. This association does not occur in the truncated NF2-16 protein nor in a mutant NF2-17 protein lacking C-terminal sequences. These data indicate that merlin has a unique mechanism of tumor suppression, inhibiting cell proliferation via self-association.

Cross-talk between different transcription factors, notably between the glucocorticoid receptor and AP-1, has been discovered more than 10 years ago: a bona fide transcription factor, without apparent need for its own direct DNA contact, influences the activity of another transcription factor. Recent experiments have added interesting aspects: in addition to major insights into the mechanism of cross-talk, it is now clear that the cross-talk ability of glucocorticoid receptor is essential for mouse development, while the activation of target promoters carrying a glucocorticoid response element (GRE), is surprisingly, dispensable for survival under animal house conditions. Interestingly, the cross-talk function is responsible for almost all regulatory actions of cortisol in the immune system. It is possible that the two functions of the glucocorticoid receptor can be activated separately by specific ligands. Future goals will be to define whether adverse effects of long-term corticosteroid treatment, e.g. osteoporosis, joint necroses, metabolic effects, can be ascribed to GRE-target gene activation and thus be dissociated from the desirable actions in the treatment e.g. of autoimmune disease.

Proteolytic cleavage of the extracellular domain of CD44 from the surface of cells has been observed recently in different cell types. In cell culture supernatants of human melanoma cell lines a 70 kDa soluble CD44 protein (solCD44) was detected at concentrations of 250-300 ng/ml. Protease inhibitor studies revealed that serine proteases and metalloproteases are involved in the cleavage of CD44 from the surface of melanoma cells. To analyse a possible function of soluble CD44 a human malignant melanoma cell line was stably transfected with cDNAs encoding either wild type soluble CD44s or mutated forms with defective HA binding properties (CD44sR41A and CD44sR150A/R154A). Soluble CD44s almost completely inhibited hyaluronic acid binding by melanoma cells, whereas soluble CD44 mutated in the HA binding domain had no effect. When cultivated on hyaluronic acid, melanoma cell proliferation was induced by 30% for both the parental and the control transfected cells. This increase in proliferation was blocked completely in solCD44s-secreting transfectants, whereas solCD44sR41A and solCD44sR150A/R154A-secreting cells again showed hyaluronic acid-induced cell proliferation. These cell lines were subcutaneously injected into MF1 nu/nu mice to compare their growth as tumors in vivo. Compared to tumors derived from parental and control transfected cells, we observed a dramatic reduction of primary tumor growth with solCD44s expressing MM cells. Transfectants expressing solCD44s mutated in the HA binding domain in contrast developed fast-growing primary tumors. These results provide strong evidence that direct solCD44 interactions with hyaluronic acid interfere competitively with processes induced by hyaluronic acid binding to surface CD44. Autocrine, or drug-induced secretion of solCD44 by human melanoma cells may thus exert potent antitumoral effects in vivo.

The neu/erbB2 protooncogene is overexpressed in numerous human cancers and is mutationally activated in N-ethyl-N-nitrosourea (ENU)-induced rodent tumors of the Schwann cell lineage. We investigated whether expression of activated neu in Schwann cells is sufficient to initiate their immortalization and transformation. Clones of embryonic dorsal root ganglia cells infected with a retrovirus bearing activated neu (NID cells) were selected based on their expression of Schwann cell-specific markers. Compared to embryonic Schwann cells infected with a virus encoding empty vector, we found that NID cells have altered shapes and disorganized cytoskeletons, grow in the absence of growth factors required for normal Schwann cell survival and proliferation, and can be repeatedly passaged. Furthermore, NID cells are invasive in an in vitro matrix invasion assay and form metastatic tumors when injected into syngeneic animals. The neu-induced growth and invasive phenotypes could be reversed by drugs that inhibit Ras and Src activity. Interestingly, later stage Schwann cells infected with activated neu failed to become immortalized. These findings indicate that constitutive activation of erbB2 is sufficient to initiate the immortalization and transformation of immature Schwann cells, and support the notion that Schwann cells have particular developmental stages during which they are susceptible to immortalizing and transforming events.

Using subtractive immunization to identify cell surface epitopes expressed in a metastasis-specific fashion on cells of the rat MT-W9 mammary carcinoma model, we generated a monoclonal antibody called M-N#1. This antibody binds specifically to metastasizing cells of the MT-W9 series and also to certain other metastasizing rat mammary carcinoma cell lines. We demonstrate that the M-N#1 antibody recognizes a fucosylated N-glycosyl sugar modification, and furthermore show that the epitope specificity of the M-N#1 antibody is for blood group antigen B subtypes 2, 3 and 4 with slight cross-reactivity with blood group antigen A subtype 2. The expression of these carbohydrate epitopes on MT-450 cells is functionally important, because the M-N#1 antibody efficiently inhibits MT-450 tumour growth in spontaneous metastasis assays. These results suggest that expression of the subtypes of blood group antigen B recognized by the M-N#1 antibody does not directly participate in the metastatic cascade but rather confers a growth or survival advantage on the tumour cells.