Biochemical mechanisms of tumor invasion and metastasis

Cancer invasion and metastases is a complex multi-step process. In order for a tumor cell to successfully traverse all the steps of this process and initiate a metastatic colony, it must express the right combination of gene products. Such gene products may include proteins which regulate cell interaction with the basement membrane and cell motility. Tumor cells attach to the basement membrane glycoprotein laminin via the cell surface laminin receptor. The human laminin receptor was purified and molecularly cloned. The level of laminin receptor mRNA in a variety of human carcinoma cells correlated with the number of laminin receptors on the surface of these cells. Following attachment to the basement membrane, the tumor cell next secretes proteases which may degrade type IV collagen. A genetic linkage between type IV collagenase secretion and metastases was collagen. A genetic linkage between type IV collagenase secretion and metastases was studied using our new genetic system for inducing metastases by employing the ras oncogene. Following attachment and local proteolysis, the third step of invasion is tumor cell motility. We have isolated a tumor cell autocrine motility factor (AMF). This factor is secreted by the tumor cells and binds to a cell surface receptor, resulting in a profound (greater than 100 x) stimulation of cell locomotion. AMF may play a major role in the autonomous invasive behavior of tumor cells.     

Possible role of oxygen radicals in cell transformation by diethylstilbestrol and related compounds

Diethylstilbestrol (DES) and four derivatives, viz. tetrafluoro-DES,3'-hydroxy-DES, Z, Z-dienestrol and hexestrol, were examinedfor their abilities to form superoxide radicals and to induceDNA strand breaks in the presence of horseradish peroxidase/hydrogenperoxide metabolism in a cell-free system. Furthermore, theinduction of strand breaks by these compounds was tested inSyrian hamster embryo (SHE) cells in vitro. Formation of superoxideradicals could be demonstrated by reduction of nitro blue tetrazoliumfor DES but not for its derivatives. With isolated superhelicalDNA, induction of strand breaks in the presence of Fe³⁺ wasobserved for DES, tetrafluoro-DES and 3’-hydroxy-DES,while hexestrol and Z, Z-dienestrol were ineffective. In SHEcells, alkaline elution technique showed that DNA strand breakswere induced by DES and all derivatives tested, although onlyat cytotoxic concentrations. It is concluded that DES, underconditions of peroxidative metabolism, can give rise to superoxidegeneration and DNA strand breaks, and that these events mayplay a role in the process of DES-induced cell transformation.     

Depression in patients referred for psychiatric consultation A need for a new diagnosis

The authors analyzed 329 referrals for psychiatric consultation from medical and surgical wards. They found depression to be the most prevalent diagnosis (34%), with Major Depression being the most common DSM-III subtype (49%). Depression was predominant in the elderly (p<0.05), in women (p<0.05), and in patients with a high degree of psychosocial stressors (p<0.01). There were significant differences among the DSM-III subtypes of depression in some of these correlates. The authors emphasize the importance of DSM-III in differentiating among the subtypes of depression in referred patients but they suggest the need for a new diagnosis for depression in the physically ill.     

Gastrointestinal function testing model using a new laryngopharyngeal pH probe (Restech) in patients after Ivor-Lewis esophagectomy

BACKGROUNDThere is no established correlation between 24-h esophageal pH-metry (Eso-pH) and the new laryngopharyngeal pH-monitoring system (Restech) as only small case series exist. Eso-pH was not designed to detect laryngopharyngeal reflux (LPR) and Restech may detect LPR better. We have previously published a dataset using the two techniques in a large patient collective with gastroesophageal reflux disease. Anatomically, patients after esophagectomy were reported to represent an ideal human reflux model as no reflux barrier exists. AIMTo use a human reflux model to examine our previously published correlation in these patients. METHODSPatients after Ivor Lewis esophagectomy underwent our routine follow-up program with surveillance endoscopies, computed tomography scans and further exams following surgery. Only patients with a complete check-up program and reflux symptoms were offered inclusion into this prospective study and evaluated using Restech and simultaneous Eso-pH. Subsequently, the relationship between the two techniques was evaluatedRESULTSA total of 43 patients from May 2016 - November 2018 were included. All patients presented with mainly typical reflux symptoms such as heartburn (74%), regurgitation (84%), chest pain (58%), and dysphagia (47%). Extraesophageal symptoms such as cough, hoarseness, asthma symptoms, and globus sensation were also present. Esophageal 24-hour pH-metry was abnormal in 88% of patients with a mean DeMeester Score of 229.45 [range 26.4-319.5]. Restech evaluation was abnormal in 61% of cases in this highly selective patient cohort. All patients with abnormal supine LPR were also abnormal for supine esophageal reflux measured by conventional Eso-pH. CONCLUSIONPatients following esophagectomy and reconstruction with gastric interposition can ideally serve as a human reflux model. Interestingly, laryngopharyngeal reflux phases occur mainly in the upright position. In this human volume-reflux model, results of simultaneous esophageal and laryngopharyngeal (Restech) pH-metry showed 100% correlation as being explicable by one of our reflux scenarios.     

Role of secretory events in modulating human neutrophil chemotaxis.

The relationship between neutrophil polymorphonuclear leukocyte (PMN) locomotion and the exocytosis of neutrophil cytoplasmic granules was studied by assessing these processes in cells migrating through micropore filters and by measuring the effects of degranulating stimuli on PMN chemotaxis, orientation, adhesiveness, and ability to bind the chemoattractant f-Met-Leu-[3H]Phe. Studies of cells migrating through cellulose nitrate filters indicated that concentrations of f-Met-Leu-Phe optimal for exocytosis were greater than those optimal for chemotaxis and actually inhibited cell migration. In other studies incubation of PMNs with concentrations of secretagogues causing exocytosis of 30% or greater PMN lysozyme increased cell adhesiveness and inhibited chemotaxis. PMNs that had secreted more than 30% lysozyme appeared round, did not orient in a gradient of chemoattractant, and were capable of significantly less f-Met-Leu-[3H]Phe binding than were control cells. The decreased binding of f-Met-Leu-Phe was not associated with hydrolysis of chemotactic peptide by washed cells, although peptide hydrolysis was caused by cell products secreted extracellularly after vigorous exocytosis. In contrast, when only 10--15% cellular lysozyme was released f-Met-Leu-Phe binding was enhanced significantly and there was no depression of chemotaxis. The data indicate limited exocytosis of intracellular granule contents is associated with increased availability of PMN cehmotactic factor receptors. Vigorous exocytosis is associated with inactivation of chemotactic responsiveness related to increase cell adhesiveness, decreased PMN binding of chemotactic factors, and to hydrolysis of chemoattractants by factors secreted extracellularly.     

Growth factors and stromal support generate very efficient retroviral transduction of peripheral blood CD34+ cells from Gaucher patients

We have achieved high-efficiency gene transfer into nonmobilized peripheral blood (PB) CD34+ cells from patients with Gaucher's disease using a clinically acceptable retroviral supernatant transduction protocol. In our studies, bone marrow (BM) and PB CD34+ cells were transduced using a high titer (10(8) particles/mL) retroviral supernatant once a day for 4 consecutive days in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF), with or without an irradiated allogeneic BM stromal layer. The growth factors alone resulted in 29% +/- 10% gene transfer of PB CD34+ clonogenic cells in contrast with 71% +/- 17% gene transfer efficiency using stroma with the growth factors; a 2.5-fold increase. The increase in gene transfer efficiency was less prominent when BM CD34+ cells were used (40% +/- 16% without and 57% +/- 8% with stroma, a 1.5-fold increase). The overall transduction efficiency of both PB and BM CD34+ cells was lower when the cells were transduced over a stromal cell layer without added growth factors. The combination of IL-3, IL-6, and SCF with stroma transduced 75% of primitive long-term culture initiating cells (PB LTC- ICs) in comparison with 34% of LTC-ICs when IL-3, IL-6, and SCF were used without stromal support. Using this clinically acceptable supernatant/cytokines/stroma transduction protocol, correction of the glucocerebrosidase (GC) deficiency in the progeny cells of PBLTC-ICs from Gaucher's-disease patients has been accomplished. Efficient transduction of the PB CD34+ cells using this transduction protocol may allow repeated delivery of "GC-corrected" hematopoietic stem and progenitor cells to Gaucher's-disease patients.     

A force-based protein biochip

A parallel assay for the quantification of single-molecule binding forces was developed based on differential unbinding force measurements where ligand-receptor interactions are compared with the unzipping forces of DNA hybrids. Using the DNA zippers as molecular force sensors, the efficient discrimination between specific and nonspecific interactions was demonstrated for small molecules binding to specific receptors, as well as for protein-protein interactions on protein arrays. Finally, an antibody sandwich assay with different capture antibodies on one chip surface and with the detection antibodies linked to a congruent surface via the DNA zippers was used to capture and quantify a recombinant hepatitis C antigen from solution. In this case, the DNA zippers enable not only discrimination between specific and nonspecific binding, but also allow for the local application of detection antibodies, thereby eliminating false-positive results caused by cross-reactive antibodies and nonspecific binding.     

Chemoattractants stimulate degradation of methylated phospholipids and release of arachidonic acid in rabbit leukocytes.

When rabbit peritoneal leukocytes were treated with chemoattractants such as fMet-Leu-Phe, an apparent decrease of [3H]methyl incorporation into the lipid fraction from L-[methyl-3H]methionine was observed. This decrease was a result of increased degradation of methylated phospholipids, not of decreased synthesis. Chemotactic peptides did not affect the metabolism of the phospholipids in which [methyl-14C]choline was incorporated. The disappearance of the [3H]methyl group was associated with the release of [1-14C]arachidonic acid from phospholipids prelabeled with these compounds. These findings suggested the activation by chemoattractants of phospholipase A2, an enzyme that removes an unsaturated fatty acid from phospholipids. The order of potency of chemoattractants for the stimulated degradation of phospholipids was in good agreement with that for chemotaxis. Mepacrine (quinacrine) and hydrocortisone inhibited and a phorbol ester enhanced both chemotaxis and phospholipase A2 activity. These results, taken together, suggest close association of the metabolism of methylated phospholipids with chemotaxis in rabbit peritoneal leukocytes.