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We tested the efficacy of gefitinib in the prevention of HER2/neu-mediated breast cancer development in BALB-NeuT transgenic mice. Oral administration of gefitinib to female transgenic mice from 5 to 14 weeks of age reduced tumor multiplicity from 9.6 +/- 0.82 to 0.58 +/- 1.1 (83%). We observed a decrease in the number and size of lobules and lobular nodules in treated mice with a reduction in the overall disease burden per gland. Normal duct development in the mammary glands was not affected by gefitinib. The development of acinic cell carcinoma in the parotid glands of these animals was also reduced coincident with decreased stromal involvement during progression. Gefitinib eliminated phosphorylation of HER2 and HER3 and signaling through MAPK and Akt in lobular hyperplasias and carcinomas. At the same time MAPK activity and cytokine production in splenocytes and lymph nodes was increased in gefitinib-treated animals coincident with an increase in lymph node size. Delaying gefitinib treatment until mammary glands exhibited atypical lobular hyperplasias reduced efficacy. These studies demonstrate the critical role of HER2 signal transduction in the onset and progression of HER2/neu-dependent breast cancer and suggest a role for specific inhibitors to prevent the outgrowth of early hyperplastic disease.

Understanding the role of signal transduction in regulating pathways responsible for cell growth, survival and apoptosis is critical for cancer therapy. We developed and characterized a HER2/neu and Fas overexpressing cell line (BNT.888 ACA2) from a salivary gland adenocarcinoma that arose in a HER2/neu transgenic mouse. We evaluated the effects of Iressa on signal transduction networks downstream of the activated HER2 and the impact on proliferation, cell cycle and apoptosis. Iressa treatment diminished phosphorylation of the HER2/neu and EGFR. Phosphorylation of STAT-3 also decreased and mitogenic signaling through the MAPK pathways was greatly reduced. Cyclin D1 levels decreased, and cells were arrested in G0 and failed to enter S-phase because of hypophosphorylation of Rb and to traverse the G2M checkpoint because of degradation of cyclin B1. Cytostasis occurred within 48 hr at 250-500 nM Iressa. Levels of proapoptotic factors (bim and bax) increased and levels of antiapoptotic factors (bcl-2 and bcl-xL) decreased in a dose-dependent manner. Higher doses of Iressa diminished phosphorylation of Akt slightly, but failed to induce apoptosis. Fas antibody was a potent agonist of apoptosis. Pretreatment with Iressa (1 microM, 24 hr) greatly enhanced Fas-mediated apoptosis as determined by Annexin V binding, cleavage of caspase-3 and PARP. Augmentation of apoptosis was associated with increased Fas expression and membrane localization. Iressa pretreatment increased bid activation, cleavage of caspases -3, -9 and -12 and stress signaling via c Jun. These data showing that Iressa induces cytostasis and primes the extrinsic (Fas) and intrinsic (mitochondrial and endoplasmic reticulum) apoptotic pathways should lead to the development of novel therapeutic targets and strategies., (2006 Wiley-Liss, Inc.)

The INK4A gene, localized to human chromosome 9p21, encodes p16INK4A, a tumor suppressor that functions at least in part through the inhibition of CDK4, a cyclin-dependent kinase encoded by a gene at 12q13. To examine INK4A gene alterations in uncultured samples of osteosarcoma and the relationship between INK4A and CDK4 alterations, we analyzed the INK4A and CDK4 genes in 87 specimens from 79 patients. INK4A deletion and CDK4 gene amplification were determined by quantitative Southern blot analysis. INK4A exon 2 was screened for mutation by polymerase chain reaction and single-strand conformational polymorphism analysis. Methylation at the CpG island in INK4A, associated with loss of p16INK4A expression, was assessed by Southern blot analysis using methylation-sensitive restriction enzymes. INK4A deletion (4/55) or rearrangement (1/55) was found in 5 of 55 cases. No INK4A exon 2 point mutations and methylation were detected. CDK4 gene amplification was found in 6 of 67 samples, but not in tumors with INK4A alteration. Amplification analysis of other genes at 12q13 (GLI, CHOP, HMGI-C and MDM2) in these 6 cases supports the view that CDK4 and MDM2 are independent targets for amplification, with variable amplification of the intervening region containing HMGI-C. Of 46 patients studied for both INK4A alterations and CDK4 amplification, the tumors in 22% contained one or the other. The prevalence of these alterations, in conjunction with the reported inactivation of RB in up to 80% of cases, suggests that genetic lesions deregulating the G1 to S cell cycle checkpoint may be an almost constant feature in the pathogenesis of osteosarcoma.

We report on a young male patient with an overgrowth syndrome, who had normal birth weight. He had a number of manifestations typical of the Weaver syndrome (WS), such as advanced bone age, peculiar craniofacial appearance, and camptodactyly. He also showed severe mental and speech retardation and demineralisation of the bones of the hands and feet. The latter can be considered as unreported manifestations of WS, or the patient could represent an example of a new WS-like syndrome.

We describe a dominant point mutation in the COL1A1 gene causing extremely severe osteogenesis imperfecta (OI type II/III) which was detected in the dermal fibroblasts of a proband, diagnosed by ultrasonography at 24 weeks of gestation. Type I collagen secretion was reduced and pro alpha 1(I) chains were overmodified. The mutation was localised in one COL1A1 allele by chemical cleavage of mismatched bases in normal cDNA/proband's mRNA heteroduplexes, and identified by cloning and sequencing. A G-to-A transition which causes the substitution of Gly-415 with serine in the alpha 1(I) triple helical domain was found. The same mutation was detected in the father's spermatozoa and lymphocytes. Mosaicism in the father's germline explains the occurrence in the family of two additional OI pregnancies, which were documented by X-ray and ultrasound investigations.