Kinase-deficient neu proteins suppress epidermal growth factor receptor function and abolish cell transformation.

p185c-neu and epidermal growth factor receptor (EGFR) associate into an active heterodimer, and overexpression of these two receptors leads to a transformed phenotype. However, the association of EGFR and kinase-deficient Neu proteins (point mutant N757 or cytoplasmic domain deletion mutant N691stop) results in a defective or inactive heterodimeric complex. In this report we explore the biological consequences of heterodimerization between EGFR and wild-type (WT) or kinase-deficient mutant Neu proteins in living cells. We show that co-expression of EGFR and kinase-deficient Neu proteins abolished the synergistic transformation and tumorigenicity. Moreover, the normal responses of EGFR to ligand were significantly suppressed, e.g., loss of EGF-dependent transformation, reduced rate of receptor endocytosis and turnover, diminished DNA synthesis, and decreased EGF binding affinity. These results provide the first evidence that kinase-deficient Neu proteins suppress normal EGFR function and display a dominant negative mutant phenotype. Together with the stimulatory effects observed in cells forming active heterodimers, these studies provide a role for heterodimerization of EGFR and Neu/c-erbB2 in interreceptor activation and synergistic signaling which may be responsible for the transition from normal receptor function into oncogenesis.

Expression of c-erbB-2 in in situ and in adjacent invasive ductal adenocarcinomas of the female breast.

Several lines of evidence have demonstrated that expression of the c-erbB-2 gene product contributes to the malignant phenotype. We and others have determined that c-erbB-2 is substantially expressed in most ductal in situ carcinomas of the comedo type, but not in other patterns of ductal carcinoma in situ or in atypical ductal hyperplasia of the breast. In the present investigation, by immunohistochemistry we inquired whether invasive ductal adenocarcinomas retained the c-erbB-2 expression status of the in situ carcinomas from which they derived. Of twelve specimens containing both cribriform/micropapillary in situ and derivative invasive adenocarcinomas in the same section, all tumor cells were negative for c-erbB-2 expression. In thirteen in situ carcinomas of the comedo type, with identifiable invasive components, ten had definite c-erbB-2 expression, and in every case there was comparable c-erbB-2 protein staining of in situ and invasive components; in three of these ten cases the staining in the in situ component tended to be more intense. These findings imply that a significant proportion of invasive mammary adenocarcinomas expressing c-erbB-2 protein is derived from ductal in situ carcinomas of the comedo type.

Analysis of c-erbB-2 protein expression in conjunction with DNA content using multiparameter flow cytometry.

Breast cancer is a leading cause of cancer death among women. Factors useful for determining the prognosis of breast cancer include axillary lymph node involvement, tumor size, hormonal receptor status, nuclear grade, and relative DNA content. The c-erbB-2 protooncogene is amplified in 10-40% of primary breast tumors, as well as in breast cancer cell lines; where it is amplified there is increased expression of its product. We have investigated the DNA content and c-erbB-1 protein expression in tumor cell lines and in breast cancer patient specimens by multiparameter flow cytometry. The study was enabled by the discovery that both cellular integrity and c-erbB-2 antigen reactivity were preserved in cells and tissues following fixation in 70% ethanol. We demonstrate that flow cytometric analysis of c-erbB-2 expression in populations of ethanol-fixed tumor cells is a reliable and sensitive quantitative method that correlates well with previously documented semiquantitative techniques. This is a feasible method for analyzing archived clinical samples, and further allows correlations between c-erbB-2 levels and other cellular parameters. Additionally, this method detects abnormal populations not identified by DNA content analysis alone. Further studies utilizing this approach are necessary to evaluate the prognostic value of this oncoprotein in human breast cancer.

Strongyloides stercoralis: parasitological, immunological and pathological observations in immunosuppressed dogs.

Four specific pathogen-free, two to three-month-old, laboratory-raised beagles were infected with Strongyloides stercoralis and monitored by thrice-weekly faecal examinations. Larvae were not detected in the faeces after the 11th week. During the 14th week, daily oral prednisolone (2.2 mg/kg) was given. This led to weak recrudescence of the infection. During the 21st week, the daily dose of prednisolone was doubled and thereafter two of the four dogs developed severe hyperinfective strongyloidiasis with their adult worm populations markedly exceeding the number of larvae inoculated, as did one dog which remained asymptomatic. One dog died during the 15th week, i.e., before there was time for a marked increase in the parasite population. Three of the four dogs provided evidence of disseminated infection with adult worms occurring in ectopic sites. Parasite-specific cellular and humoral immune responses were monitored weekly. All dogs developed a significant rise in specific IgM titres lasting until the fifth week. From the fourth week, all dogs had detectable parasite-specific IgG antibody levels that persisted throughout the experiment in spite of the immunosuppression. Strongyloides-induced lymphocytic responses in vitro were demonstrated in three dogs from the second to the fifth week of infection, but returned to pre-infection levels thereafter. All animals were necropsied. Relevant pathological lesions were found in the small intestine, the colon and the lungs. Larvae were seen in such ectopic sites as mesenteric nodes and prostate. The observations provide further evidence that canine S. stercoralis infections can be manipulated to reproduce with great fidelity the parasitological and clinico-pathological events occurring in human disseminated strongyloidiasis.

Strongyloides stercoralis: hyperinfection in immunosuppressed dogs.

Hyperinfective strongyloidiasis involving the threadworm , Strongyloides stercoralis, is well known in humans and primates. Although this nematode also frequently parasitizes dogs, canine hyperinfective strongyloidiasis has not been reported. To determine whether a fulminant pattern of nematode development can occur in dogs, and to test the S. stercoralis/dog system for suitability as a model for human hyperinfective and disseminated strongyloidiasis, five canine infections with a dog-derived strain of S. stercoralis were monitored by the quantitative recovery of larvae from feces. Even 3-month-old pups controlled their initial infections successfully, the number of larvae excreted declining to near zero in 90 days. Immunosuppressive treatment with prednisolone, prednisolone and azathiaprine , or niridazole resulted in a rapid return to former or greater intensities of infection, as judged by larval output. Only first stage ( rhabditiform ) larvae were passed in the feces, although third stage (filariform) larvae occurred in the intestinal contents of dogs when they were examined at necropsy. In 3 of the 5 dogs, the adult worm recovery exceeded the inoculated dose greatly and, in one of these, adults and rhabditiform larvae were found in distant, extraintestinal sites. In the remaining 2 of the 5 dogs, the adult worm population was less than the inoculated dose, but, in both, the infection was terminated by the host's death before hyperinfection could have developed. The observations demonstrate that autoinfection occurs in dogs infected with S. stercoralis and that, if it is allowed to continue for a sufficiently long time in immunosuppressed hosts, massive hyperinfection, and even disseminated infection, may occur. This spectrum of increasingly invasive parasitism closely resembles strongyloidiasis in humans. Therefore, the S. stercoralis/dog system has excellent potential as a model for human hyperinfective and disseminated strongyloidiasis.