Role of p27Kip1 and Cyclin-Dependent Kinase 2 in the Proliferation of Non-Small Cell Lung Cancer

The cell cycle is governed by a family of cyclin-dependent kinases (Cdks). Cdk2 forms a functional complex with cyclin E and plays a pivotal role in the regulation of G1/S transition. Cdk2 activity is negatively regulated by interactions with inhibitors. p27^Kip1, one of the most potent inhibitors of Cdk2, was recently identified as a powerful negative prognostic marker in non-small cell lung cancer as well as in colorectal and breast cancer. In the present study, the expression of p27 and Ki-67 antigen in nonneoplastic and cancerous lung tissues was determined by immunohistochemistry. After establishing that the antibody-measured p27 labeling index was a good reflection of the level of p27 expression measured by Western blotting, we show that p27 labeling index is decreased in cancerous lung tissues, compared with nonneoplastic lung tissues, and exhibits a significant inverse relation to the proliferation marker Ki-67 antigen, detected with monoclonal antibody MIB-1. Consistent with these data, all cancerous lung tissues showed enhanced degradation activity of p27 compared with nonneoplastic lung tissues and, in addition, increased levels of the phosphorylated form of Cdk2, as determined with Western blot analysis. The H1 histone kinase activity associated with Cdk2 was also increased in non-small cell lung cancers. Statistical analysis showed that proliferative activity as measured by MIB-1 labeling index was highly correlated with Cdk2 activity (r = 0.767, P < 0.0015). These results suggest that p27 and Cdk2 may play an important role in the proliferation of non-small cell cancer.     

Two distinct antigenic types of the polysaccharide chains of Helicobacter pylori lipopolysaccharides characterized by reactivity with sera from humans with natural infection

We have purified lipopolysaccharides (LPS) from 10 Helicobacter pylori clinical isolates which were selected on the basis of chemotype and antigenic variation. Data from immunoblotting of the purified LPS with sera from humans with H. pylori infection and from absorption of the sera with LPS indicated the presence of two distinct epitopes, termed the highly antigenic and the weakly antigenic epitopes, on the polysaccharide chains. Among 68 H. pylori clinical isolates, all smooth strains possessed either epitope; the epitopes were each carried by about 50% of the smooth strains. Thus, H. pylori strains can be classified into three types on the basis of their antigenicity in humans: those with smooth LPS carrying the highly antigenic epitope, those with smooth LPS carrying the weakly antigenic epitope, and those with rough LPS. Sera from humans with H. pylori infection could be grouped into three categories: those containing immunoglobulin G (IgG) antibodies against the highly antigenic epitope, those containing IgG against the weakly antigenic epitope, and those containing both specific IgGs; these groups made up about 50%, less than 10%, and about 40%, respectively, of all infected sera tested. In other words, IgG against the highly antigenic epitope were detected in more than 90% of H. pylori-infected individuals with high titers. IgG against the weakly antigenic epitope were detected in about 50% of the sera tested; however, the antibody titers were low. The two human epitopes existed independently from the mimic structures of Lewis antigens, which are known to be an important epitope of H. pylori LPS. No significant relationship between the reactivities toward purified LPS of human sera and a panel of anti-Lewis antigen antibodies was found. Moreover, the reactivities of the anti-Lewis antigen antibodies, but not human sera, were sensitive to particular alpha-L-fucosidases. The human epitopes appeared to be located on O-polysaccharide chains containing endo-beta-galactosidase-sensitive galactose residues as the backbone. Data from chemical analyses indicated that all LPS commonly contained galactose, glucosamine, glucose, and fucose (except one rough strain) as probable polysaccharide components, together with typical components of inner core and lipid A. We were not able to distinguish between the differences of antigenicity in humans by on the basis of the chemical composition of the LPS.     

Incidental prostatic carcinoma: Morphometry correlated with histological grade

Summary The histological grades of prostatic carcinoma, as defined by Gleason, were correlated with three methods of morphometry in 254 step-sectioned prostates obtained at autopsy. The variables studied were 1) the number of tumours in each prostate; 2) bilaterality and 3) tumour volume. Each characteristic yielded a statistically significant correlation with histological grade. The strongest correlations were obtained using tumour volume. These autopsy studied help to explain the inconsistent results obtained from morphometric analyses of surgical material, and lend support to the Gleason system as a means of predicting tumour behavior.Supported in part by research contracts PH 64-10, NCI-72-3213, N01-CP-53521; Grant R01-CA-33644; and the Grant-in-Aid for Cancer Research (33) from the Ministry of Health and Welfare     

Allergen-induced biphasic bronchoconstriction in rats

The development of an allergic bronchoconstriction model in rats is described. In actively sensitized Donryu strain rats, there was a remarkable biphasic increase in airway resistance within 10 min after antigen challenge on day 9 to day 21. The increase in airway resistance, correlated with the IgE titer and the dose of antigen, was inhibited by disodium cromoglycate (DSCG) or by aminophylline. This bronchoconstriction was remarkably blocked by methysergide (25 and 100 micrograms/kg) while pyrilamine inhibited it partially at the same dose. Serotonin (greater than 30 micrograms/kg) but not histamine (less than 1,000 micrograms/kg) induced a bronchoconstriction. FPL-55712 (1,10 mg/kg) inhibited it significantly. The content of thromboxane B2 (TxB2) in plasma increased during the bronchoconstriction while the content of peptide-leukotrienes (p-LTs) in plasma did not increase significantly. OKY-046 inhibited not only allergic bronchoconstriction but also the increase in TxB2 levels in plasma. The late phase of the bronchoconstriction was more susceptible to OKY-046. In conclusion, this model seems to be useful for the screening of antiasthma drugs because of a relationship with the dose of antigen, IgE titer and the susceptibility to an antiallergic drug or a bronchodilator. It is demonstrated that the major part of this allergic bronchoconstriction depends on serotonin, and it is also suggested that thromboxane A2 may play an important role in the late phase of the bronchoconstriction.     

Simple,rapid, and accurate determination of deletion mutations by automated dna sequencing of heteroduplex fragments of the adenomatous polyposis coli (APC) gene generated by PCR amplification

Germline mutations in patients with familial adenomatous polyposis were analyzed by polymerase chain reaction (PCR) amplification of the adenomatous polyposis coli gene. PCR products from heterozygous patients for deletions of this gene formed four distinct bands on polyacrylamide gel electrophoresis. The four fragments were subsequently purified and both strands of each fragment were directly sequenced, using an automated DNA sequencer and the same primers as those for PCR amplification. It was found that the two slower migrating fragments were “bulge” heteroduplexes, while the other two were homoduplexes made up of two wild-type strands and two deletion-mutant strands, respectively. The sites of deletions in the adenomatous polyposis coli gene could be exactly determined in four of the five patients. In an attempt to identify deletion-carriers of familial adenomatous polyposis at the presymptomatic stage, a family study was also carried out, and two children were found to have the same mutations as those of their affected parents. The direct sequencing of heteroduplex fragments generated during PCR amplification is a potentially useful method for detecting mutations of not only the adenomatous polyposis coli gene but also many other genes of genetic diseases. © 1993 Wiley-Liss, Inc.     

Social isolation stress augments angiogenesis induced by colon 26-L5 carcinoma cells in mice

We have previously shown that tumor necrosis factor-α (TNF-α), which is an important angiogenesis-related factor, was over-secreted in male BALB/c mice under social isolation stress as compared with the control, and closely associated with a remarkable elevation of tumor invasion and metastasis of colon 26-L5 carcinoma cells. In the present study, we explored the effect of isolation stress on the angiogenesis caused by colon 26-L5 carcinoma cells in vivo and in vitro. Social isolation lead to the enhancement of tumor growth after intrahepatic implantation with a fragment of colon 26-L5 tumor. Angiogenic response (number of vessels oriented towards tumor mass) and tumor growth (size) were significantly increased in the socially isolated mouse relative to that in the group-housed mice. Furthermore, higher protein level of hepatic TNF-α was found in the stressed mice than that in the control. Expression of mRNA for vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were also elevated in the tumor regions and liver tissues of the stressed mice in comparison with that in group-housed mice. On the other hand, hepatic sinusoidal endothelial (HSE) cells treated with TNF-α exhibited a marked promotion of the migration, invasion, expression of mRNA for matrix metalloproteinase (MMP)-9, and tube-like formation, but no cytotoxicity against the cells in vitro. The above data suggest that the social isolation stress augmented the tumor-induced angiogenesis probably by up-regulating the angiogenesis-related factors, including TNF-α, VEGF and HGF, and consequently mediating the functions of endothelial cells such as migration, invasion, and tube-like formation.     

Role of tight junctions of polarized epithelial MDCK cells in the replication of herpes simplex virus type 1

Before completion of polarization, Madin-Darby canine kidney (MDCK) cells showed high infectivity and progeny production of herpes simplex virus type 1 infection. After polarization or formation of tight junctions, the infectivity and virus replication in MDCK cells was restricted significantly. The disruption of tight junctions by depletion of Ca²⁺ resulted in increasing virus infectivity and productivity. Mechanical disruption of tight junctions by scratching the cell monolayers with injection needle allowed markedly the replication of HSV-I in the cells aligned along the injured area. In polarized MDCK cells the progeny were released preferentially from the apical surface of the cells. These data suggest that because polarized MDCK cells mimic the epithelial cell layers, this cell line is helpful for determining the factors which regulate viral transmission in the human body. © Wiley-Liss, Inc.     

Anaplastic carcinoma of the thyroid gland. An ultrastructural study on four cases.

Four cases of anaplastic carcinoma of the thyroid, composed of one small cell carcinoma and three giant cell carcinomas, were studied with electron microscope. In the case of small cell carcinoma, fine cytoplasmic interdigitations and junctional complex between apposing cytoplasmic membranes of neighbouring tumor cells and a few microlumina within tumor cell clusters surrounded by well-defined basal lamina were seen. In the cases of giant cell carcinoma, occasional cytoplasmic interdigitations as well as desmosomal structures were detected even in tumor cells markedly pleomorphic and anaplastic. Abundant cytoplasmic organelles including profiles of Golgi apparatus, rough endoplasmic reticulum and a few mitochondria were seen in the cytoplasm of tumor cell of all four cases. Of interest to note was that all giant cell carcinomas demonstrated evidences of fairly well differentiated tumor within anaplastic carcinoma, indicating probable pre-existing either benign or malignant epithelial neoplasm more differentiated, with its subsequent anaplastic transformation. Findings in the present study support an assumption that these anaplastic tumors are derived from the follicular epithelium of the thyroid gland. In addition, it can be said that tumor cells of the small cell carcinoma provide evidences suggesting functional differentiation of carcinoma cells to a certain extent, yet unable to produce thyroglobulin.