Absence Of Contact Inhibition Research Articles

MAP17, a ROS-dependent oncogene

MAP17 is a small 17 kDa non-glycosylated membrane protein previously identified as being overexpressed in carcinomas. Breast tumor cells that overexpress MAP17 show an increased tumoral phenotype with enhanced proliferative capabilities both in the presence or the absence of contact inhibition, decreased apoptotic sensitivity, and increased migration. MAP17-expressing clones also grow better in nude mice. The increased malignant cell behavior induced by MAP17 is associated with an increase in reactive oxygen species (ROS) production, and the treatment of MAP17-expressing cells with antioxidants results in a reduction in the tumorigenic properties of these cells. The MAP17-dependent increase in ROS and tumorigenesis relies on its PDZ-binding domain because disruption of this sequence by point mutations abolishes the ability of MAP17 to enhance ROS production and tumorigenesis. MAP17 is overexpressed in a great variety of human carcinomas, including breast tumors. Immunohistochemical analysis of MAP17 during cancer progression demonstrates that overexpression of the protein strongly correlates with tumoral progression. Generalized MAP17 overexpression in human carcinomas indicates that MAP17 can be a good marker for tumorigenesis and, especially, for malignant progression.

MAP17 enhances the malignant behavior of tumor cells through ROS increase

Tumorigenesis occurs when the mechanisms involved in the control of tissue homeostasis are disrupted and cells stop responding to physiological signals. Therefore, genes capable of desensitizing tumoral cells from physiological signals may provide a selective advantage within the tumoral mass and influence the outcome of the disease. We undertook a large-scale genetic screen to identify genes able to alter the cellular response to physiological signals and provide selective advantage once tumorigenesis has begun. We identified MAP17, a small 17 kDa non-glycosylated membrane protein previously identified by differential display being over-expressed in carcinomas. Tumor cells that over-express MAP17 show an increased tumoral phenotype with enhanced proliferative capabilities both in presence or absence of contact inhibition, decreased apoptotic sensitivity and increased migration. MAP17-expressing clones also grow better in nude mice. The increased malignant cell behavior induced by MAP17 are associated with an increase in reactive oxygen species (ROS) production, and the treatment of MAP17-expressing cells with antioxidants results in a reduction in the tumorigenic properties of these cells. Treatment of melanoma cells with inhibitors of Na+-coupled co-transporters lead to an inhibition of ROS increase and a decrease in the malignant cell behavior in MAP17-expressing clones. Finally, we show that MAP17-dependent ROS increase and tumorigenesis are dependent on its PDZ-binding domain, since disruption of its sequence by point mutations abolishes its ability to enhance ROS production and tumorigenesis. Our work shows the tumorigenic capability of MAP17 through a connection between Na+-coupled co-transporters and ROS.

Hepatitis C Virus Core Protein Transforms Murine Fibroblasts by Promoting Genomic Instability

The oncogenic potential of hepatitis C virus (HCV) core protein has been demonstrated, but the precise mechanism of cell transformation triggered by HCV core is still unclear. This study shows that constitutive expression of HCV core protein (core) in NIH 3T3 murine fibroblasts triggers malignant transformation. At the preneoplastic stage, clones that expressed HCV core constitutively demonstrated genomic instability seen as disruption of the mitotic spindle cell checkpoint leading to increased ploidy. Transformation was completed by the loss of DNA and resistance to apoptosis induced by serum starvation. Simultaneously, cells acquired a capacity for anchorage independent growth and absence of contact inhibition. Inoculation of these transformed cells into severe combined immune deficiency (SCID) mice led to formation of solid core-expressing tumors. Transformation and tumorigenicity of core-expressing cell lines coincided with a 5- to 10-fold repression of endogenous p53 transactivation. Thus, long-term HCV core expression alone is sufficient for complete transformation of immortal fibroblasts that can then induce tumors in a susceptible host. This data suggests that malignant transformation by HCV core may occur through primary stress, induction of genomic instability, and further HCV core-induced rescue of surviving mutated cells.

Establishment and characterization of human neuroblastoma and ganglioneuroblastoma cell lines.

The establishment in culture and characterization of 4 human neuroblastoma (NB) cell lines and 1 human ganglioneuroblastoma cell line are described. Each cell line fulfilled at least 2 of 4 criteria for malignant or transformed cells: viz., subcultured more than 70 times, high saturation density with absence of contact inhibition, population-doubling time within a range of 10-40 h, and tumour formation in nu/nu mice. Each cell line also fulfilled at least 2 of 3 criteria for neuroblastoma cells: viz., humoral and cell-mediated immune reactivity toward NB-associated cell-surface antigen, intracellular storage and extra-cellular secretion of catecholamines, and characteristic neuroblast and ganglion-cell morphology.

Establishment of a new cell line (NRC-12) derived from a human renal cell carcinoma and its characteristics (author's transl)

A permanent established cell line (NRC-12) derived from a human renal cell carcinoma is described. These cells have been maintained for 30 months and over 140 in vitro passages. The growth of these epithelial cells was characterized by the absence of contact inhibition and the doubling time was constant at 48 hours. Electron microscopic study showed the localized nucleus and prominent microvilli resembling the brush border-like structures of the renal cell carcinoma on the opposite side of the nucleus. Histrogical examination of the heterotransplant into the cheek pouches of a golden hamster verified that the nature of the cells was identical to clear cell adenocarcinoma, resembling the original tumor of the patient. Chromosomal analysis revealed the presence of two hypotetraploid stemlines (86 and 88) and a secondary constriction on chromosome A3 of all cells.

Characterization of a cell line from human transitional cell cancer of the urinary tract.

A cell line designated 253J was established from a biopsy specimen of retroperitoneal metastasis in a man with multiple transitional cell cancers of the urinary tract. The cells in early and late passages were small and epithelial and had a doubling time of 48 hours. The growth of these cells in culture was characterized by the absence of contact inhibition as evidenced by multilayering. There were virus-like particles in both the original surgical specimen and in cells in culture. The particles were in cytoplasmic vesicles, free in the cytoplasm, or outside both normal and degenerating cells. These particles, varying in size from 30 to 130 nm, had an electron-dense nucleoid and were bounded by a unit membrane. Karyotypic analysis showed the presence of a hyperdiploid stemline with persistent marker chromosomes and chromosomes with multiple rearrangements resulting in unusual G and C banding patterns. The malignant nature of the line was verified by 1) the production of transplantable tumors in immune-depressed hamsters after inoculation with 253J cell suspension and 2) the growth of 253J cells in agar. These cells, in culture over 24 months, have been subcultured over 60 times and continue to retain their epithelial morphology and malignancy.

Two distinct types of enhancement of galactose uptake into hamster cells: tumor-virus transformation and hexose starvation.

Enhancement of hexose uptake seems well correlated with transformation of cell cultures by tumor viruses and the absence of contact inhibition. Enhancement of sugar uptake has also been observed as a result of hexose starvation. Both types of enhancement can clearly be demonstrated in cultures of hamster cells when uptake of (14)C-labeled galactose is monitored after 10 or 20 min. The profiles of accumulation products are strikingly different. In cultures of hamster NIL cells transformed with polyoma virus much of the (14)C is accumulated as UDPhexose. Untransformed cells accumulate galactose-l-phosphate as well as UDPhexose. Hexose-starved cells show enhanced uptake of galactose; however, this marked enhancement was only observed in NIL cultures close to contact inhibition. The novel and common feature seen in hexose-starved cells when incubated briefly with (14)C-labeled galactose is the occurrence of a marked accumulation of [(14)C]UDPglucuronic acid at the expense of UDPhexose. The ratio [(14)C]UDPglucuronic acid/UDPhexose in cultures fed glucose or galactose was invariably low (0.15-0.2) regardless of the presence or absence of contact inhibition. 20 hr of hexose starvation invariably changed this ratio by a factor of 10 or more, due to accumulation of UDPglucuronic acid. This result was also observed in cultures transformed with polyoma virus. The presence of 3-O-methylglucose in the growth medium did not alter the typical "sugar starvation pattern" (i.e., the UDPglucuronic acid/UDPhexose ratio averaged 1.7). Enhancement of galactose uptake by hexose starvation was very pronounced in NIL cultures that were close to contact inhibition, but was not a prominent feature in the polyoma-transformed cultures. The transformed cells grown on glucose or galactose growth medium showed the usual enhanced rate of uptake of galactose as compared with nontransformed near-confluent cultures that had been fed hexose. The polyoma-induced enhancement showed none of the features characteristic of hexosestarved cells.

Ultrastructural modifications of intercellular junctions between tumor cells.

During recent years increasing evidence has accumulated on the participation of surface phenomena in the regulation of cell growth and cell movement in multieellular organisms. Studies on the nature of cell to cell interactions have proved to be relevant in elucidating the complex mechanisms involved in normal morphogenetie cell movements and in the establishment and maintenance of neoplastic growth. Cellular interactions have been studied at best advantage in culture systems, where poorly understood regulatory mechanisms operate mostly through intercellular contact (1). In normal cell cultures, the establishment of social behavior patterns appears to depend mainly on the inhibitory effects of intercellular contact on the motility and the growth of the cell. In tumor cells, the absence of contact inhibition of movement and division has been related to the unrestricted cell growth and the invasive properties shown by tumor cells in vivo. Although the importance of surface phenomena in cell growth regulation has been recognized, little is known on the structural features of the cellular interactions of adjacent normal or transformed cultured cells. Regions of close membrane apposition have been described between cultured fibroblasts (2); in fibroblasts of developing tissues a different type of contact has been described as "attachment sites" (3). The study of normal intercellular relationships between cultured cells is facilitated by the use of an in situ embedding technique which preserves the characteristics of the culture (4). When sections are cut parallel to the surface of a normal monolayered culture, the parallel disposition of the cells, their fusiform shape, and the regions of cell attachment are readily seen (Fig. 1). If normal cultures are cut following a plane perpendicular to the surface of the culture, the monolayer character becomes apparent; overlapping is minimal and when present involves only thin cytoplasmic processes. In contrast, similarly oriented sections of adenovirus 12-transformed hamster embryo cells show several layers of cells piled on top of each other (Fig. 2).