Edith S. Robbins

Tumor invasion through the human amniotic membrane: Requirement for a proteinase cascade

To understand the role of proteinases in tumor invasion, the effects of inhibitors of metallo-, serine-, and cysteine-proteinases on this process were studied using 125I-iododeoxyuridine-labeled B16/BL6 cells grown on human amnion basement membrane. Cellular invasion was quantitated by measuring the radioactivity associated with the amniotic membrane after the B16/BL6 cells on the basement membrane were removed by lysis followed by scraping. The results obtained with proteinase inhibitors showed that inhibitors of collagenase and plasmin prevented invasion of the amnion. Tissue invasion was also blocked by antiurokinase antibodies. On the contrary, cysteine-proteinase inhibitors and anti-tissue plasminogen activator antiserum were ineffective. Mersalyl, a compound known to activate collagenase, stimulated invasion under conditions where plasmin formation or activity were inhibited. Evidence for the role of a plasminogen activator-plasmin-collagenase activation cascade in B16 invasion is provided.

Stage-specific surface antigens expressed during the morphogenesis of vertebrate forms of Trypanosoma cruzi

The origin of Trypanosoma cruzi slender and broad forms found in the circulation of the mammalian host has remained obscure and, unlike what has been proposed for African trypanosomes, no precise form-function relationship has been ascribed to them. We show here that parasites circulating in the blood of infected animals display a high degree of polymorphism. Around 10% of the forms found circulating in mice during the acute phase of infection were amastigotes, and the other 90% included slender and broad trypomastigotes and intermediate forms between amastigotes and trypomastigotes. Slender trypomastigotes, from blood or cell culture, undergo extracellularly morphological rearrangements in which the parasites become gradually broader and transform into amastigotes. By scanning electron microscopy a progressive internalization of the flagellum and reorganization of the cell shape in a helical fashion were observed in parasites undergoing transformation. After 48 hr of extracellular incubation the parasite population consisted exclusively of amastigotes with a short protruding flagellum. The morphological changes were associated with the expression of different surface antigens defined by monoclonal antibodies: the trypomastigote-specific antigens Ssp-1 (a 100-120-150-Mr glycoprotein), Ssp-2 (a 70-Mr glycoprotein), Ssp-3 (undefined), and Ssp-4, an amastigote-specific surface antigen. Ssp-4 was also detected on intracellular amastigotes (in vitro and in vivo). We conclude that trypomastigotes are programmed to develop into amastigotes whether or not they enter cells, and that the differentiation can occur in the blood of the vertebrate host. These findings raise some questions regarding conventional views on the life cycle of T. cruzi.

Roles of uroplakins in plaque formation, umbrella cell enlargement, and urinary tract diseases.

The apical surface of mouse urothelium is covered by two-dimensional crystals (plaques) of uroplakin (UP) particles. To study uroplakin function, we ablated the mouse UPII gene. A comparison of the phenotypes of UPII- and UPIII-deficient mice yielded new insights into the mechanism of plaque formation and some fundamental features of urothelial differentiation. Although UPIII knockout yielded small plaques, UPII knockout abolished plaque formation, indicating that both uroplakin heterodimers (UPIa/II and UPIb/III or IIIb) are required for plaque assembly. Both knockouts had elevated UPIb gene expression, suggesting that this is a general response to defective plaque assembly. Both knockouts also had small superficial cells, suggesting that continued fusion of uroplakin-delivering vesicles with the apical surface may contribute to umbrella cell enlargement. Both knockouts experienced vesicoureteral reflux, hydronephrosis, renal dysfunction, and, in the offspring of some breeding pairs, renal failure and neonatal death. These results highlight the functional importance of uroplakins and establish uroplakin defects as a possible cause of major urinary tract anomalies and death.

Regulation of the Drosophila apoptosome through feedback inhibition

Apoptosis is induced by caspases, which are members of the cysteine protease family. Caspases are synthesized as inactive zymogens and initiator caspases first gain activity by associating with an oligomeric complex of their adaptor proteins, such as the apoptosome. Activated initiator caspases subsequently cleave and activate effector caspases. Although such a proteolytic cascade would predict that a small number of active caspases could irreversibly amplify caspase activity and trigger apoptosis, many cells can maintain moderate levels of caspase activity to perform non-apoptotic roles in cellular differentiation, shape change and migration. Here we show that the Drosophila melanogaster apoptosome engages in a feedback inhibitory loop, which moderates its activation level in vivo. Specifically, the adaptor protein Apaf-1 lowers the level of its associated initiator caspase Dronc, without triggering apoptosis. Conversely, Dronc lowers Apaf-1 protein levels. This mutual suppression depends on the catalytic site of Dronc and a caspase cleavage site within Apaf-1. Moreover, the Drosophila inhibitor of apoptosis protein 1 (Diap1) is required for this process. We speculate that this feedback inhibition allows cells to regulate the degree of caspase activation for apoptotic and non-apoptotic purposes.

The variability in radon decay product bronchial dose

Abstract The dose to cell nuclei in the bronchial airways from the inhalation of 222 Rn decay products was calculated using the most recent data for all biological and physical parameters. The major factors that influenced the bronchial dose were the median aerosol diameter, the breathing rate, the unattached fraction, and the depth of the cell nuclei in the airway. The best estimate of the basal cell dose was 6 mGy/WLM in both homes and mines, with a range of 3–15 mGy/WLM. The dose to mucous cell nuclei was about 40% greater.

Radon and Leukemia in the Danish Study: Another Source of Dose

An epidemiologic study of childhood leukemia in Denmark (2,400 cases; 6,697 controls) from 1968 to 1994 suggested a weak, but statistically significant, association of residential radon exposure and acute childhood lymphoblastic leukemia (ALL). The Danish study estimated a relative risk (RR) = 1.56 (95% CI, 1.05-2.30) for a cumulative exposure of 1,000 Bq m-3 y. For an exposure duration of 10 y their RR corresponds to a radon concentration of 100 Bq m-3. There are two dose pathways of interest where alpha particles could damage potential stem cells for ALL. One is the alpha dose to bone marrow, and two is the dose to bronchial mucosa where an abundance of circulating lymphocytes is found. Compared with an exposure of about 1 mSv y-1 from natural external background, radon and decay products contribute an additional 10 to 60% to the bone marrow equivalent dose. The other pathway for exposure of T (or B) lymphocytes is within the tracheobronchial epithelium (BE). Inhaled radon decay products deposit on the relatively small area of airway surfaces and deliver a significant dose to the nearby basal or mucous cells implicated in human lung cancer. Lymphocytes are co-located with basal cells and are half as abundant. Using a 10-y exposure to 100 Bq m-3, our dose estimates suggest that the equivalent dose to these lymphocytes could approach 1 Sv. The relatively high dose estimate to lymphocytes circulating through the BE, potential precursor cells for ALL, provides a dose pathway for an association.

Platelet Blockade of Particle Absorption from the Peritoneal Surface of the Diaphragm

Abstract Particulate material is absorbed from the peritoneal cavity via respiration-induced gaps between mesothelial cells on the peritoneal surface of the diaphragm and is then collected into diaphragmatic and retrosternal lymph vessels. The effect of platelets on this process was examined by (i) comparing the appearance of these lymph vessels after intraperitoneal (ip) injection of Radiopaque or carbon particles in control and experimental rats receiving, respectively, an ip injection of either platelet-rich (PRP) or platelet-poor (PPP) plasma or saline solution 1 hr prior to the injection of particles; and by (ii) inspecting lymph vessels on the pleural side of 20 freshly excised pieces of rabbit or dog diaphragm (secured pleural side down, to the mouth of a suction flask) 5 min after application of India ink with either PRP or PPP (or saline) to the peritoneal surface. In each case injection or application of PRP either blocked or markedly restricted particle absorption. Electron microscopic (EM) observations indicate that platelets block absorption of particles by plugging gaps between adjacent mesothelial cells.

A biokinetic model for 222Rn gas distribution and alpha dose in humans following ingestion

A biokinetic model for absorption and distribution of [sup 222]Rn in the body following ingestion in drinking water was developed. The rate parameters for the model are derived from historical human data reported in experiments of Harley et al. (1958) and Hursh et al. (1965). The biokinetic model fits the human data of Hursh et al. well for whole body retention following ingestion of [sup 222]Rn in water on an empty stomach. The equivalent dose to stem cells for stomach and colon cancer is estimated to be 1.6 [times] 10[sup [minus]9] Sv Bq[sup [minus]1]. The model calculations yield 4 and 3 calculated stomach and colon cancers from continuous intake of water at the estimated US average of 5.5 Bq L[sup [minus]1]. 16 refs., 3 figs., 2 tabs.

Radon carcinogenesis: risk data and cellular hits

Abundant epidemiological data are now available (2008) on the human lung cancer response for lifetime radon gas exposure to residential concentrations of 100 Bq m(-3), equal to 22 working level months over 40 y. We combined published pooled epidemiological data and dosimetric calculations of alpha particle hits to target basal or mucous cell nuclei in bronchial epithelium. This yields an estimate that about 10,000 basal nuclei (target) cell hits per cm2 per person over a lifetime are involved in radon-related lung cancer. The DNA target cell area (cross section) for a hit is about 2 bp. The present epidemiology indicates that 1000 persons need to be exposed to this hit rate for observable cancers to be detected. The mechanism proposed is that the extensive prior DNA damage in smokers, followed by alpha particle damage to a critical site in checkpoint genes, accounts for the greater lung cancer response in smokers.

A biological model for lung cancer risk from 222Rn exposure

Abstract A model for lung cancer risk from 222 Rn exposure was derived based on the following assumptions: 1) a cell nucleus in division (cycling) in the bronchial epithelium is the target for carcinogenesis; and 2) risk is proportional to the hit nucleis ability to subsequently cycle. The model development required two steps. First, the number of cycling basal cell nuclei that can be traversed by an alpha particle following an exposure to 1 working level month (WLM) was calculated. Second, the model utilized measured cycling rates of basal nuclei in normal bronchial epithelium, to calculate the dividing population as a function of time post exposure. The basal nuclei cycling rates, from preliminary data, decreased with age with a half life of about 15 y. The shape of the model, as a function of time since exposure was compared with underground miner risk data, and the agreement was good.