Involvement of growth factor receptors in the mammalian UVC response.

Irradiation of HeLa cells with short-wavelength ultraviolet light (UVC) induces the modification and activation of the preexisting transcription factors c-Fos-c-Jun (AP-1) and TCF/Elk-1, as well as the protein synthesis independent transcriptional activation of the c-fos and c-jun genes. This response to UVC is mediated via obligatory cytoplasmic signal transduction, involving Ras and Raf, Src, and MAP kinases. The UVC response is inhibited by prior down-modulation of growth factor receptor signaling upon growth factor prestimulation, by suramin (an inhibitor of receptor activation) or by expression of a dominant negative epidermal growth factor (EGF) receptor mutant. These data suggest the involvement of several growth factor receptors in the UVC response. Indeed, UVC induces the suramin-inhibitable immediate tyrosine phosphorylation of the EGF receptor.

The mammalian UV response: mechanism of DNA damage induced gene expression.

DNA damage inducing treatment of cultured mammalian cells triggers the activation of transcription factors and the prolongation of the half life of p53. As the earliest event detectable in the nucleus (5 min), AP-1 (c-Jun/c-Fos) is post-translationally modified. Triggering this early event and triggering subsequent transcription factor dependent processes requires extra-nuclear components of signal transduction such as Src, Ras, Raf-1 and MAP-2 kinase. Recent efforts have concentrated on examining whether DNA damage or other secondary effects of the damaging agent generate the signal then passed on to transcription factors. Further, it has been studied whether a pathway of reverse signalling exists that originates in the nucleus and reaches the cell surface. At the cell surface the UV induced signalling chain can be interrupted experimentally. Beyond this step DNA damage and signal transduction induced by phorbol esters and growth factors merge and reach the nuclear proteins through common components.

Damage to DNA by UV light and activation of transcription factors.

Bacteria react to irradiation with short wave length UV (UVC) by mounting a rescuing response which involves the synthesis of proteins engaged in DNA repair, replication and mutagenesis. We analyse here an analogous response shown by mammalian cells in culture and present experimental evidence for the chain of events induced by UV irradiation that leads to enhanced gene expression. Available results suggest that the UV induced signal cascade depends on damage to DNA and also involves components located at the plasma membrane, such as src, ras and raf. These components, upon activation by UV, signal into the cell's nucleus, thereby activating transcription factors which control the activity of UV responsive genes.

UV-induced activation of AP-1 involves obligatory extranuclear steps including Raf-1 kinase.

Irradiation of cells with ultraviolet light (UV) leads to modifications of c-Jun resembling those elicited by phorbol esters or oncogenes, and to enhanced transcription of AP-1-dependent genes. The UV-induced signal also triggers activation of Raf-1 and MAP-2 kinases. A dominant-negative Raf-1 kinase mutant strongly interferes with both phorbol ester and UV-induced AP-1 activation, indicating obligatory involvement of identical components in cytoplasmic signal transduction. Thus, from a presumably nuclear site of energy absorption, a signal needs to be transmitted to the cytoplasm in order to achieve activation of a nuclear transcription factor. Further, signals elicited from different primary sites merge prior to or at the level of activation of Raf-1 kinase.

A novel T-cell trans-activator that recognizes a phorbol ester-inducible element of the interleukin-2 promoter.

The interleukin 2 (IL-2) gene promoter is recognized by several cell-type-specific and ubiquitous transcriptional regulators that integrate information transmitted by various signaling systems leading to IL-2 production and T-cell activation. Using a combination of transfection, protein-DNA binding, and in vitro transcription methods, we have discovered the novel T-cell-specific transcriptional activator TCF-1 (for T-Cell Factor-1), which recognizes a T-cell-specific response element (TCE) located within the IL-2 promoter. Although the TCE is similar in sequence to a consensus NF kappa B site, several criteria indicate that TCF-1 is distinct from NF kappa B. However, like NF kappa B, TCF-1 activity is induced by phorbol esters and other T-cell activators.

Induction of urokinase activity and malignant phenotype in bladder carcinoma cells after transfection of the activated Ha-ras oncogene.

In order to characterize further the previously observed induction of a highly metastatic phenotype in mouse bladder carcinoma cells by Ha-ras transfection, we studied production of plasminogen activator, in vitro invasiveness, and the potential for lung colonization of these cells. The parent carcinoma cells produced predominantly tissue-type plasminogen activator. Out of 13 clones of ras-transfected cells tested, 8 secreted quantitatively elevated levels of plasminogen activator (up to 3.5-fold) as compared to the control transfectants. The plasminogen activator activity in cell lysates was maximally increased 3-fold, the surface-associated activity increased 2.5-fold. The secreted plasminogen activator of cloned ras-transfected cells was characterized to be predominantly of the urokinase type (71.3% compared to 20.5% with the parental BL cells). Thus, in addition to the quantitative augmentation of plasminogen activator production and secretion in a large fraction of the ras-transfected cell population, a significant qualitative shift from tissue-type to urokinase-type has been observed. In addition, ras-transfection augmented the capacity of the cells for invasion into Matrigel in a double-filter in vitro assay as well as their ability to colonize the lungs of syngeneic animals. These malignant properties of the transfected cells might be responsible for their highly metastatic behaviour induced by ras transfection.

Induction of the metastatic phenotype by transfection of the nuclear oncogene p53: increases in cytoplasmic diacylglycerol levels and reduction in class I major histocompatibility antigen expression are not sufficient to explain the changes in metastatic capacities.

Transfection of the oncogene encoding the nuclear protein p53 into a low-metastatic mouse carcinoma cell line resulted in enhanced metastatic capabilities in clones that showed increased p53 protein expression [Pohl J, Goldfinger N, Radler-Pohl A, Rotter V, Schirrmacher V (1988) Mol Cell Biol 8:2078-2081]. This effect seemed neither to be due to increase in cytoplasmic diacylglycerol levels nor to reduced cell-surface expression of class I major histocompatibility antigens.

A model to account for the effects of oncogenes, TPA, and retinoic acid on the regulation of genes involved in metastasis.

We have postulated that signals from the microenvironment can induce shifts in tumor cell phenotypes and that microenvironmental factors are therefore important for cancer metastasis. In this article we expand on this hypothesis and propose a model to explain (a) how extracellular signals can lead to changes in tumor phenotypes, and (b) how cytoplasmic oncogenes, which influence signal transducing pathways as well as nuclear oncogenes regulating gene expression via DNA binding transacting factors, might affect metastatic competence.

Selective enhancement of metastatic capacity in mouse bladder carcinoma cells after transfection with DNA from liver metastases of human colon carcinoma.

To identify sequences associated with a metastatic phenotype, DNA fragments isolated from 2 separate human colon carcinoma metastases were transfected into a mouse bladder carcinoma cell line together with the neoR gene as selectable marker. It was found that bulk populations of neomycin resistant cells carrying these human sequences caused more metastases in syngeneic mice than did control cells transfected with calf thymus DNA. Cells isolated from metastases retained the highly metastatic phenotype when transferred to secondary hosts.

Oncogene expression in related cancer lines differing in metastatic capacity.

Various murine tumor cell lines with different metastatic capacities were tested in vitro for oncogene expression, especially of the p21-Ha-ras protein. Small differences were seen in the expression of several distinct oncogenes in the case of a high metastatic lymphoma variant (ESb) and its low metastatic parental line (Eb). In one instance we observed a 30-fold Ha-ras gene amplification in a metastasis-derived cell line from a spontaneous mouse mammary carcinoma. In spite of this amplification we did not find an increased p21 expression in these cells.

p53 increases experimental metastatic capacity of murine carcinoma cells.

Transfection of a cloned p53 gene into a murine bladder carcinoma cell with a low metastatic capacity led to elevated levels of p53 protein in clonal transfectants. After intravenous inoculation into syngeneic mice, p53-transfected clones showed significantly increased metastatic potential in comparison with control transfectants. The observed change did not seem to be due to a change in growth potential per se since the cell lines showed similar growth properties in vitro.

Analysis of metastatic competence of mouse bladder carcinoma cells after transfection with activated Ha-ras or N-ras oncogenes.

Transfection of the Ha-ras oncogene into a low metastatic epithelial cell line resulted in the acquirement of significantly increased metastatic capacity. This alteration in metastatic competence of a carcinoma line in a syngeneic system seemed to be a selective change and was not affected by parameters such as tumor latency period or local tumor growth. Transfection of the selection marker vectors with normal cellular DNA or with the N-ras gene did not lead to significantly increased metastatic capacity. Analysis of metastatic variants after oncogene transfection and in vivo selection showed integration of N-ras, but not of Ha-ras oncogenes. A possible role for the Ha-ras oncogene in the initial steps of metastasis will be discussed.