Neoadjuvant chemoradiotherapy with concurrent cisplatin/5-fluorouracil is associated with increased pathologic complete response and improved survival compared to carboplatin/paclitaxel in patients with locally advanced esophageal cancer.

Trimodal therapy consisting of neoadjuvant chemoradiation followed by esophagectomy has become the standard of care in North America for locally advanced esophageal cancer. While cisplatin/5-fluorouracil has been a common concurrent chemotherapy regimen since the 1980s, its utilization has declined in recent years as the Chemoradiotherapy for Oesophageal Cancer Followed by Surgery Study (CROSS) trial regimen of carboplatin/paclitaxel has become widely adopted. The efficacy of the CROSS regimen compared to alternate chemotherapy choices, however, has rarely been evaluated when each is used as a component of a trimodal treatment approach. The aim of this study is to report our institutional experience with these two concurrent chemotherapy regimens at a specialized esophageal cancer center.We performed an Institutional Review Board-approved retrospective review of a prospectively maintained institutional foregut registry from a single National Cancer Institute-designated cancer center. Esophageal cancer patients who completed trimodal therapy with a chemotherapy regimen of either carboplatin/paclitaxel or cisplatin/5-fluorouracil were identified and divided into groups based on their chemotherapy regimens. Multivariable logistic regression was used to analyze pathologic complete response rates, while the Kaplan-Meier and Cox proportional hazards models were utilized to evaluate recurrence-free and overall survival. Analytical models were adjusted for age, clinical stage, radiation dose, histologic subtype (adenocarcinoma vs. squamous cell carcinoma), and time interval from completion of neoadjuvant therapy to surgery.One hundred and forty-two patients treated between January of 2000 and July of 2015 were identified as meeting inclusion criteria. Of this group, 87 had received the CROSS regimen of carboplatin/paclitaxel, while 55 had completed cisplatin/5-fluorouracil. Multivariable analysis demonstrated that the cisplatin/5-fluorouracil.group had an increased odds of pathologic complete response (odds ratio = 2.68, 95% confidence interval, P = 0.032), as well as significantly improved recurrence-free survival (hazard ratio = 0.39, 95% confidence interval 0.21-0.73, P = 0.003) and overall survival (hazard ratio = 0.46, 95% confidence interval 0.24-0.87, P = 0.016), compared to the carboplatin/paclitaxel group.Concurrent chemotherapy with cisplatin/5-fluorouracil in locally advanced esophageal cancer is associated with higher rates of pathologic complete response and improved recurrence-free and overall survival compared to the CROSS regimen of carboplatin/paclitaxel. This suggests that, for select patients, alternate neoadjuvant chemotherapy approaches, such as cisplatin/5-fluorouracil, merit reconsideration as potential primary treatment choices in the management of this highly morbid disease.

Microtubular crystals in mammalian cells.

Periwinkle alkaloids in very low concentrations cause an intracytoplasmic sequestration of microtubule protein in the form of symmetrical, microtubular bodies. These crystals, which may measure up to 8 micro in length, appear within 30 min in L-strain fibroblasts in vitro, but they increase in incidence and size with time of exposure to the alkaloids. Similarly, if exposed to these compounds, human leukocytes in vitro contain identical crystalline structures. Neither colchicine nor puromycin prevents the formation of these bodies; the latter compound, however, retards crystal growth.

Spectrin rearrangement early in erythrocyte ghost endocytosis.

The endocytic vacuoles induced in white ghosts were found to be depleted of spectrin and therefore it was proposed that they arose from spectrin-free areas in the erythrocyte membrane. To follow changes in spectrin distribution during endocytosis, affinity-purified rabbit antispectrin antibodies were produced. Quantitative techniques were developed for the use of a highly specific 125I-F(ab')2 antispectrin, and these showed that before the appearance of vacuoles, as assessed by phase microscopy, there was a reproducible decrease in immunoreactive spectrin. To determine whether this spectrin decrease represented a local or diffuse spectrin loss or a spectrin rearrangement, morphologic studies were undertaken using transmission electron microscopy on samples treated with rabbit antispectrin and ferritin-conjugated goat anti-rabbit immunoglobulin. These studies showed that endocytosis was preceded by the creation of extensive spectrin-free areas separated by discrete spectrin-containing zones. Pretreatment of ghosts with alkaline phosphatase blocked all forms of endocytosis and prevented the creation of spectrin-free areas. Therefore, it is proposed that under the impetus of endocytosis inducers, phosphorylated spectrin is redistributed so that spectrin-free zones are created, and that endocytic vacuoles form and fuse in spectrin-free areas.

Basal lamina formation by cultured microvascular endothelial cells.

The production of a basal lamina by microvascular endothelial cells (MEC) cultured on various substrata was examined. MEC were isolated from human dermis and plated on plastic dishes coated with fibronectin, or cell-free extracellular matrices elaborated by fibroblasts, smooth muscle cells, corneal endothelial cells, or PF HR9 endodermal cells. Examination of cultures by electron microscopy at selected intervals after plating revealed that on most substrates the MEC produced an extracellular matrix at the basal surface that was discontinuous, multilayered, and polymorphous. Immunocytochemical studies demonstrated that the MEC synthesize and deposit both type IV collagen and laminin into the subendothelial matrix. When cultured on matrices produced by the PF HR9 endodermal cells MEC deposit a subendothelial matrix that was present as a uniform sheet which usually exhibited lamina rara- and lamina densa-like regions. The results indicate that under the appropriate conditions, human MEC elaborate a basal lamina-like matrix that is ultrastructurally similar to basal lamina formed in vivo, which suggests that this experimental system may be a useful model for studies of basal lamina formation and metabolism.

Absorption of intact protein molecules across the pulmonary air-tissue barrier.

The majority of heterologous serum albumin and globulin molecules introduced into the pulmonary alveoli of dogs are absorbed into the circulatory system antigenically intact. This function of the alveoli has both physiologic and pathologic importance.

Human polymorphonuclear leukocytes: demonstration of microtubules and effect of colchicine.

Microtubules are demonstrable in mature human polymorphonuclear leukocytes, even after prolonged incubation in vitro. These organelles are not seen after treatment with colchicine, which has various functional effects on these cells but does not inhibit phagocytosis.

Vinblastine and griseofulvin reversibly disrupt the living mitotic spindle.

Using polarized light we have studied the effects of various mitotic poisons on mitotic spindles of living Pectinaria oocytes; we have studied fixed specimens with phase and electron microscopy. Vinblastine caused attrition and eventual disappearance of spindle structure as rapidly as did colcemid, and subsequent recovery from this treatment was at least as fast as that from colcemid. Griseofulvin, however, was easily the best agent for rapid, reversible, and repeated dissolution of the spindle. Agents that arrest metaphase may act on nondividing cells by interfering with the organization of other gelated structures.

Induction in vitro of microtubular crystals by vinca alkaloids.

The addition of vinblastine or vincristine to solutions of pure microtubule protein or to supernatants from high-speed centrifugation of rabbit-brain homogenates results in the formation of a fine precipitate. Examination of this precipitate by electron microscopy reveals ordered structures with areas of ladder-like configuration.

Age-dependent changes in proteins of Drosophila melanogaster.

Several molecular theories of aging postulate that there are age-dependent changes in gene expression and that these changes contribute to the reduction in the viability of senescent cells. High-resolution, semiautomated, quantitative two-dimensional gel electrophoresis of many soluble proteins was used to test this hypothesis in Drosophila. Two-dimensional protein gel patterns were analyzed for each of three age groups of [(35)S]methionine-labeled adult male Drosophila melanogaster, which, except for their spermatocytes, consist entirely of fixed postmitotic cells. Seven relatively abundant polypeptides expressed in middle-aged (28-day-old) flies were absent in both young(10-day-old) and old (44-day-old) flies. Quantitative analyses of an additional 100 polypeptides were carried out by computer-assisted microdensitometry of fluorograms of the gel preparations. These analyses revealed a significant age-related heterogeneity in the quantitative distribution of radiolabel in these proteins. The data indicate that the qualitative pattern of gene expression is identical in young and old flies, but that profound quantitative changes occur in the expression of proteins during the Drosophila life-span.

Drug-induced erythrocyte membrane internalization.

In vitro erythrocyte membrane internalization, resulting in the formation of membrane-lined vacuoles, can be quantified by a radioisotopic method. A complex of (37)Co-labeled vitamin B(12) and its plasma protein binders is first adsorbed to the cell surface, and after vacuoles are formed, the noninternalized label is removed by washing and trypsin treatment. The residual radioactivity represents trapped label and can be used to measure the extent of membrane internalization. Using this method, it was found that in addition to primaquine, a group of membrane-active drugs, specifically hydrocortisone, vinblastine, and chlorpromazine can induce membrane internalization in erythrocytes. This is a metabolic process dependent on drug concentration, temperature, and pH. Vacuole formation by all agents tested can be blocked by prior depletion of endogenous substrates or by poisoning the erythrocytes with sodium fluoride and sulfhydryl blocking agents. This phenomenon resembles in some respects the previously reported membrane internalization of energized erythrocyte ghosts. It is suggested that membrane internalization is dependent on an ATP-energized state and is influenced by the balance between the concentrations of magnesium and calcium in the membrane. This study provides a basis for proposing a unifying concept of the action of some membrane-active drugs, and for considering the role of erythrocyte membrane internalization in pathophysiologic events.

Energized endocytosis in human erythrocyte ghosts.

The mechanism of endocytosis in resealed human erythrocyte ghosts was studied. The energy for endocytosis or micropinocytosis appears to be derived from Mg-ATP, and membrane internalization is preceded by activation of a membrane-associated Ca,Mg-ATPase and by the active efflux of Ca. Endocytosis, Ca,Mg-ATPase activity, and active Ca efflux all require the presence of Mg. Furthermore, these three phenomena, endocytosis, Ca,Mg-ATPase activity, and active Ca extrusion, all have a concentration dependence on Ca such that low concentrations stimulate and higher concentrations inhibit the phenomena. The optimal concentration of Ca is identical for endocytosis, active Ca efflux, and Ca,Mg-ATPase. Morphologic studies indicated that while active Ca efflux and activation of the Ca,Mg-ATPase activity occurred promptly upon onset of incubation, there was a significant time delay before endocytosis occurred, which suggests that endocytosis additionally involved a more slowly functioning mechanicochemical mechanism. Ruthenium red, a specific inhibitor of Ca,Mg-ATPase and Ca transport, inhibited endocytosis in a concentration-related manner. Prostaglandins E1 and E2 had no measurable effect on ghost endocytosis, active Ca efflux, or Ca,Mg-ATPase activity.

Gastrin releasing peptide is a selective mitogen for small cell lung carcinoma in vitro.

Human small cell lung carcinoma (SCLC) cells have been shown to contain significant levels of a bombesin-immunoreactive peptide. The 27-amino acid peptide, gastrin releasing peptide (GRP), has recently been shown to be responsible for the bombesin-like immunoreactivity found in SCLC cells. Among four lung cancer cell lines examined in vitro, GRP exhibited mitogenic activity for two SCLC subtypes, but not for a squamous carcinoma or adenocarcinoma lung cell line. The mitogenicity of the GRP molecule has been isolated to the carboxyterminal fragment, designated GRP 14-27, which is in part homologous to bombesin. The aminoterminal fragment, GRP 1-16, is no homologous to bombesin and exhibits no mitogenic activity. Thus, GRP may be an important growth regulating or autocrine factor in human SCLC.

Expression of bovine superoxide dismutase in Drosophila melanogaster augments resistance of oxidative stress.

Superoxide dismutases (SOD) play a major role in the intracellular defense against oxygen radical damage to aerobic cells. In eucaryotes, the cytoplasmic form of the enzyme is a 32-kDa dimer containing two copper and two zinc atoms (CuZn SOD) that catalyzes the dismutation of the superoxide anion (O2-) to H2O2 and O2. Superoxide-mediated damage has been implicated in a number of biological processes, including aging and cancer; however, it is not certain whether endogenously elevated levels of SOD will reduce the pathological events resulting from such damage. To understand the in vivo relationship between an efficient dismutation of O2- and oxidative injury to biological structures, we generated transgenic strains of Drosophila melanogaster overproducing CuZn SOD. This was achieved by microinjecting Drosophila embryos with P-elements containing bovine CuZn SOD cDNA under the control of the Drosophila actin 5c gene promoter. Adult flies of the resulting transformed lines which expressed both mammalian and Drosophila CuZn SOD were then used as a novel model for evaluating the role of oxygen radicals in aging. Our data show that expression of enzymatically active bovine SOD in Drosophila flies confers resistance to paraquat, an O2(-)-generating compound. This is consistent with data on adult mortality, because there was a slight but significant increase in the mean lifespan of several of the transgenic lines. The highest level of expression of the active enzyme in adults was 1.60 times the normal value. Higher levels may have led to the formation of toxic levels of H2O2 during development, since flies that died during the process of eclosion showed an unusual accumulation of lipofuscin (age pigment) in some of their cells. In conclusion, our data show that free-radical detoxification has a minor by positive effect on mean longevity for several strains.

Invasion of reconstituted basement membrane matrix by metastatic human tumor cells.

A gel-like reconstituted basement membrane matrix containing type IV collagen, laminin, entactin, nidogen, and heparan sulfate proteoglycan was used to examine the invasive properties of human HT1080 fibrosarcoma cells. Within several hours after seeding, the tumor cells initiated a random migration, leaving behind channels etched in the surface of the matrix. Eventually the channels became interconnected into a complex network. As the tumor cells proliferated, the channels became filled until the surrounding matrix was gradually dissolved. Cells then migrated outward, forming the typical disorganized cell monolayer normally observed when fibrosarcoma cells are cultured on plastic surfaces. In contrast to the fibrosarcoma cells, normal skin fibroblasts, while able to attach to the matrix, exhibited minimal migration, tracking, and invasion during the same time period. When tumor cells were seeded onto thick layers of matrix, the cells ultimately invaded downward into the matrix, leaving behind open tunnels. At the front of the invading cells, long irregular pseudopodia projected in the direction of movement. Electron microscopy demonstrated these filopodial and lamellopodial projections to directly extend into the surrounding matrix, with focal clearing of the matrix in the immediate vicinity of these invading pseudopodia. Thus, tumor cell invasion of extracellular matrices, including basal lamina, may proceed by the formation of specialized pseudopodia that not only form adhesion contacts with the matrix but also provide an efficient mechanism for the focal hydrolysis of the matrix at the site of directed cell movement.

Gastrin-releasing peptide-related peptides in a human malignant lung carcinoid tumor.

Recent immunohistochemical findings have indicated the presence of gastrin-releasing peptide in normal and pathological human lungs. Gastrin-releasing peptide is a 27-amino acid peptide isolated from porcine gut which bears considerable carboxyterminal homology with bombesin. We have characterized the gastrin-releasing peptide-like peptides present in a human malignant lung carcinoid tumor by gel chromatography and reverse-phase high-performance liquid chromatography. Our results show that this tumor did not contain bombesin; however, this tumor expressed a gastrin-releasing peptide-like compound, several amino-terminal fragments, and a carboxy-terminal fragment of gastrin-releasing peptide.

Myoglobinuria associated with herpes-group viral infections.

Two cases of myoglobinuria were associated with herpes-group viral infection (herpes simplex and Epstein-Barr). Muscle atrophy and acute renal failure were important complications. No evidence of direct invasion of muscle fibers by virus was found, nor was the severity of myoglobinuria correlated with histologic appearance of muscle.

Acromegaly and pheochromocytoma: a multiple endocrine syndrome caused by a plurihormonal adrenal medullary tumor.

A 42-yr-old man with congestive heart failure and diabetes mellitus was found to have acromegaly and a pheochromocytoma. Serum GH-releasing hormone (GHRH) levels were elevated (2.34 ng/dl; normal, less than 0.02 ng/dl), suggesting that the acromegaly was caused by ectopic secretion of GHRH. Postmortem examination revealed that the right adrenal gland contained a pheochromocytoma in which GHRH was demonstrated by immunohistochemical studies. Gel permeation chromatography combined with the use of two GHRH antisera showed that GHRH-(1-44)-NH2 was a predominant form of the hormone. When the RNA from the tumor was extracted and analyzed by Northern gel blotting, two mRNA species were identified, with transcripts corresponding to 1600 and 780 base pairs. The pituitary gland was enlarged, but no distinct adenoma was found. Diffuse and nodular hyperplasia of somatotrophs in some areas resembling adenoma was identified on histological examination. These findings indicate that GH excess accompanied by somatotroph hyperplasia and acromegaly were secondary to a pheochromocytoma which secreted not only catecholamines but also GHRH.

Acromegaly and Zollinger-Ellison syndrome secondary to an islet cell tumor: characterization and quantification of plasma and tumor human growth hormone-releasing factor.

A young woman with acromegaly and Zollinger-Ellison syndrome associated with a GH-releasing factor (GRF)- and gastrin-secreting metastatic islet cell carcinoma was studied by means of specific antisera which recognize various regions of the GRF molecule. Using specific immunohistochemical techniques, the tumor cells were shown to contain GRF, gastrin, and gastrin-releasing peptide, but not GH. During a 4-h period, plasma GRF levels averaged 5.6 +/- 1.4 ng/ml (+/- SD), while GH levels averaged 148 +/- 71 ng/ml. GH secretion was pulsatile and increased after TRH administration. GRF RIAs may be useful in establishing the diagnosis of acromegaly secondary to the ectopic secretion of GRF.

Ascorbic acid uptake by young and aged guinea pig lenses.

The dynamics of in vitro uptake of [14C]-ascorbic acid into intact lenses of young (50 days old) and old (730 days old) guinea pigs was evaluated in this study. Two-dimensional protein gel analysis of [35S]-methionine labeled proteins provided evidence that the isolated lenses were viable throughout the culture period. It was found that the in vitro uptake of ascorbate into these lenses follows similar saturation kinetics for lenses from both age groups. Moreover, the linear uptake rate was identical. Ascorbate transport was inhibited by phloretin, p-chloromercuribenzoate, insulin, glucose and ouabain; however, no inhibition was observed with 2,4-dinitrophenol or NaF. These results suggest that the lenticular ascorbate concentration is regulated by a facilitated diffusion process and it not energy-dependent. Data on ascorbate transport into lenses from aged guinea pigs provide strong evidence that the abnormally low ascorbic acid concentrations in lenses of old mammals is not likely the result of a decreased lenticular uptake ability.

Age-related changes in turnover and concentration of a subset of thorax polypeptides from Drosophila melanogaster.

Six polypeptides from Drosophila melanogaster were analyzed by two-dimensional polyacrylamide gel electrophoresis for age-related changes in protein turnover and protein concentration. The protein samples, from thoraces of 6 day (young), 26 day (middle) and 45 day (old) flies, were compared using autoradiography for [35S]methionine labeled proteins and silverstaining for unlabeled preparations. The combination of these two techniques has permitted determination of the relative turnover rate as well as the steady-state concentration of these proteins as a function of age. The autoradiographs reveal that all of the analyzed proteins decrease in turnover rate whereas the absolute protein concentrations, as determined from the silverstained gels, show a decrease for three proteins, no change for two, and an increase in one protein with age. These findings show a remarkable quantitative heterogeneity of ageing changes in proteins, but absence of alterations in the fidelity of the translation of these polypeptides.