Expression of the tumor suppressor deleted in liver cancer-1 (DLC-1) is lost in non-small cell lung (NSCLC) and other human carcinomas, and ectopic DLC-1 expression dramatically reduces proliferation and tumorigenicity. DLC-1 is a multidomain protein that includes a Rho GTPase Activating Protein (RhoGAP) domain which has been hypothesized to be the basis of its tumor suppressive actions. To address the importance of the RhoGAP function of DLC-1 in tumor suppression, we performed biochemical and biological studies evaluating DLC-1 in NSCLC. Full length DLC-1 exhibited strong GAP activity for RhoA as well as RhoB and RhoC, but only very limited activity for Cdc42 in vitro. In contrast, the isolated RhoGAP domain showed 5-to 20-fold enhanced activity for RhoA, RhoB, RhoC and Cdc42. DLC-1 protein expression was absent in six of nine NSCLC cell lines. Restoration of DLC-1 expression in DLC-1-deficient NSCLC cell lines reduced RhoA activity, and experiments with a RhoA biosensor demonstrated that DLC-1 dramatically reduces RhoA activity at the leading edge of cellular protrusions. Furthermore, DLC-1 expression in NSCLC cell lines impaired both anchorage-dependent and -independent growth, as well as invasion in vitro. Surprisingly, we found that the anti-tumor activity of DLC-1 was due to both RhoGAP-dependent and -independent activities. Unlike the rat homologue p122RhoGAP, DLC-1 was not capable of activating the phospholipid hydrolysis activity of phospholipase C-δ1. Combined, these studies provide information on the mechanism of DLC-1 function and regulation, and further support the role of DLC-1 tumor suppression in NSCLC.

DLC-1 encodes a Rho GTPase-activating protein (RhoGAP) and negative regulator of specific Rho family proteins (RhoA-C and Cdc42). DLC-1 is a multi-domain protein, with the RhoGAP catalytic domain flanked by an amino-terminal sterile ␣ motif (SAM) and a carboxyl-terminal START domain. The roles of these domains in the regulation of DLC-1 function remain to be determined. We undertook a structure-function analysis involving truncation and missense mutants of DLC-1. We determined that the amino-terminal SAM domain functions as an autoinhibitory domain of intrinsic RhoGAP activity. Additionally, we determined that the SAM and START domains are dispensable for DLC-1 association with focal adhesions. We then characterized several mutants for their ability to regulate cell migration and identified constitutively activated and dominant negative mutants of DLC-1. We report that DLC-1 activation profoundly alters cell morphology, enhances protrusive activity, and can increase the velocity but reduce directionality of cell migration. Conversely, the expression of the amino-terminal domain of DLC-1 acts as a dominant negative and profoundly inhibits cell migration by displacing endogenous DLC-1 from focal adhesions.

. The second hydroxylation is catalyzed by the 25-hydroxyvitamin D 3 -1␣-hydroxylase (1␣-hydroxylase), a mitochondrial cytochrome P450 enzyme that is the product of the CYP27B1 gene (2-6). Activity of 1␣-hydroxylase is tightly regulated through complex mechanisms that depend on the circulating levels of calcium, phosphorus, parathyroid hormone, and 1,25(OH) 2 D 3 .Mutations in the 1␣-hydroxylase gene are known to cause vitamin D-dependency rickets type I (VDDR-I) (2, 7-9). Patients afflicted with this disease are unable to maintain normal serum calcium and suffer from secondary hyperparathyroidism, rickets, and osteomalacia (10). VDDR-I is cured by administration of physiological doses of 1,25(OH) 2 D 3 (11). Physiological doses of 25-OH-D 3 are noncurative, but high dose administration can be effective (11), presumably due to the ability of 25-OH-D 3 to bind and activate the vitamin D receptor when present in vast excess.The experiments reported by Fraser and Kodicek in 1970 (12) were the first to demonstrate the kidney as the major, if not the only, tissue in which 1,25(OH) 2 D 3 is produced under normal physiological conditions. Over the next several years, extrarenal production of 1,25(OH) 2 D 3 was convincingly demonstrated in pregnant nephrectomized rats and in an anephric patient suffering from sarcoidosis (13-15). In these cases, synthesis was localized to the placenta and the sarcoid macrophages (14,16,17). Production of 1,25(OH) 2 D 3 at other sites has remained a subject of much investigation. A number of research groups have reported 1␣-hydroxylase activity in cultured cells, including those of the skin, bone, cartilage, intestine, prostate, and vascular epithelium (18 -25). Bikle et al. (26) have also reported 1,25(OH) 2 D 3 production in perfused flaps of porcine skin. Local production of 1,25(OH) 2 D 3 has been proposed to regulate cellular function and͞or differentiation in an autocrine or paracrine fashion (18,19,24,27,28), and it has been suggested that keratinocytes could supply 1,25(OH) 2 D 3 to the systemic circulation when renal production of the hormone is impaired (26,29). Production of extrarenal 1,25(OH) 2 D 3 in these experiments is not supported, however, by in vivo metabolic studies in nephrectomized nonpregnant rats. In these studies, two independent research groups were unable to detect 3 H-1,25(OH) 2 D 3 in the tissue or plasma after administering a dose of 3 H-25-OH-D 3 of high specific radioactivity (30,31). These conflicting results demonstrate a need for further investigation of the in vivo expression of the 1␣-hydroxylase. We have approached such an investigation by using gene targeting to replace the 1␣-hydroxylase coding sequence with a bacterial lacZ gene controlled by the 1␣-hydroxylase promoter. The lacZ gene codes for ␤-galactosidase, whose activity is readily detected in situ through histochemical staining with X-Gal (32). Herein we report the successful production of 1␣-hydroxylase null mice harboring the lacZ gene and present our analysis of in vivo 1␣-hydroxylase...

Summary Epidermal growth factor receptor (EGF-R) expression was assessed in 63 lung tumour samples with a monoclonal antibody (EGF-R1) by indirect immunoperoxidase staining on cryostat sections. All 15 small cell lung cancer samples were negative whereas over 80% of the 48 non small cell lung cancer stained positively.In 30 bronchial biopsies two monoclonal antibodies against the cytoplasmic part of the EGF-R were evaluated. These antibodies showed weaker staining than EGF-RI. No additional or enhanced staining as compared with EGF-R1 was observed, suggesting a lack of enhanced expression of a truncated EGF-R analogous to the v-erb-B oncogene product.Monoclonal antibodies against the EGF-R may be helpful diagnostically in differentiating small cell from non small cell lung cancer and may also be important in elucidating biological differences in primary lung cancer.