Subcellular localization of proteasomes in apoptotic lung tumor cells and persistence as compared to intermediate filaments

Failure to diagnose breast carcinoma by fine-needle aspiration (FNA) is a major obstacle to expanded use of that technique. While the majority of false negative diagnoses are due to inadequate sampling and insufficient specimens, a significant minority of false negative cases result from inaccurate interpretation of adequate material. Low grade carcinomas including lobular, tubular, low grade adenosquamous, and papillary carcinomas appear to account for many of these diagnostic errors. Careful attention to nuclear detail, monomorphism of cell population and the presence of neoplastic cells with retained cytoplasm should allow the recognition of the majority of these neoplasms as malignant by cytologic examination.     

Motile properties of vimentin intermediate filament networks in living cells

PURPOSE: To evaluate the clinical utility and morphologic accuracy of gadolinium-enhanced excretory magnetic resonance (MR) urography after low-dose diuretic injection and to correlate the results with those of conventional urography. MATERIALS AND METHODS: In 71 patients with urologic symptoms, excretory MR urography was performed after intravenous injection of 5-10 mg furosemide and, 30-60 seconds later, 0.1 mmol of gadopentetate dimeglumine per kilogram of body weight. The MR urograms were interpreted by three radiologists, who were blinded to the clinical outcome, and subsequently compared with conventional urograms. RESULTS: Injection of furosemide before contrast material led to rapid, uniform gadolinium distribution inside a sufficiently distended collecting system such that there was no excessive concentration of gadolinium in the urine. In patients with normal or moderately reduced excretory function, this effect allowed complete visualization of the urinary tract within 5-20 minutes of contrast material injection while minimizing gadolinium-related endoluminal T2* effects. The clinical course helped verify almost all MR urographic results. The MR urographic technique was significantly superior to conventional urography in the assessment of the ureters and bladder (P < .0001). Delineation of small caliceal abnormalities is still problematic. The best depiction of the pelvicaliceal system was obtained with fat-suppressed MR imaging, although it was still slightly inferior to conventional urography (P < .05). CONCLUSION: Gadolinium-enhanced excretory MR urography performed after low-dose diuretic injection is a promising and accurate alternative to conventional excretory urography for imaging the morphology of the urinary tract.     

Development of vimentin filaments in the cells of the articular disc of the rat squamosomandibular joint with age

The organochlorines, dichloro-diphenyl-trichloroethane and polychlorinated biphenyl (PCB) are pervasive environmental contaminants. Results from previous studies have been conflicting regarding the relationship between the internal dose of these organochlorine residues and breast cancer risk. To determine whether these compounds are present in breast cyst fluids and whether cyst fluid and plasma concentrations are correlated, we analyzed organochlorines in paired cyst fluid and plasma samples from 24 subjects using gas chromatography and electron capture detection. All but one of the women had a history of multiple cysts, suggesting that they were at elevated risk for future breast cancer. DDE (a metabolite of dichloro-diphenyl-trichloroethane) was present in 22 of the cyst samples and PCB was detected in 19 of the cyst samples. Organochlorine levels were more concentrated in the plasma than in breast cyst fluids. Levels of DDE in plasma were significantly correlated with those in cyst fluid (r = 0.73; P < 0.001); in contrast to PCB levels in cyst and plasma (r = 0.37; P = 0.12). Congener specific analysis of the PCBs showed that some individual congeners were preferentially excluded from or concentrated in the cyst fluid. To our knowledge, this study is the first to demonstrate that PCB and DDE are present in cyst fluids and thus in contact with the ductal epithelium of the breast. These results support the use of plasma DDE as a proxy for DDE in the target tissue in research on the role of environmental factors in breast cancer.     

Unique phosphorylation of vimentin filaments in glomerular epithelial cells in culture and in disease

Three cDNA clones for the Machado-Joseph disease gene (MJD1) were isolated, two of which have a new exon sequence and a distinct 3' terminal nucleotide sequence resulting in a new carboxyl terminal domain in the translated product. The nucleotide sequence of the other one is similar to the previously published one except for five polymorphisms, one of which is a single nucleotide substitution resulting in a change from the stop codon (TAA; allele A) to a tyrosine residue (TAC; allele C). Genetic analysis results suggest that Japanese MJD mutations are associated with allele A.     

Effect of pentobarbital on normal brain protection and on the response of 9L rat brain tumor to radiation therapy

The authors attempted to confirm published reports that pentobarbital protects against radiation-induced damage to normal rat brain, as well as enhances radiotherapeutic efficacy in a rat brain tumor model. They evaluated animal survival in 9L gliosarcoma-burdened rats that received whole-brain radiation therapy (16, 24, 32, or 40 Gy) while under intraperitoneal pentobarbital (60 mg/kg) or intramuscular ketamine (60 mg/kg) sedation. The animals were examined at autopsy to attribute death to either intracranial tumor growth or normal brain toxicity in the absence of discernible tumor. There was no difference between the two anesthesia groups regarding the survival of unirradiated animals. Radiation therapy produced a significant dose-dependent prolongation in animal survival, which was limited by the development of normal tissue toxicity at the higher doses. When compared to ketamine anesthesia, pentobarbital anesthesia appeared to offer some protection (not statistically significant) against early (but not late) toxicity at selected radiation doses. A reduction in the number of deaths from tissue toxicity suggested an increased antitumor effect, but again this was not statistically significant. Only in one case was there even a marginal significant difference (p = 0.045) between overall therapeutic efficacy in rats sedated with pentobarbital versus ketamine. While there may be a radioprotective effect of pentobarbital in rat brains without intracranial tumor, there is no conclusive evidence for either radioprotection or significant improvement of radiotherapeutic efficacy in this 9L rat brain tumor model.     

Taxol induces bcl-2 phosphorylation and death of prostate cancer cells

Treatment of prostate cancer cell lines expressing bcl-2 with taxol induces bcl-2 phosphorylation and programmed cell death, whereas treatment of bcl-2-negative prostate cancer cells with taxol does not induce apoptosis. bcl-2 phosphorylation seems to inhibit its binding to bax since less bax was observed in immunocomplex with bcl-2 in taxol-treated cancer cells. These findings support the use of the anticancer drug taxol for the treatment of bcl-2-positive prostate cancers and other bcl-2-positive malignancies, such as follicular lymphoma.     

Differential activation of p38 mitogen-activated protein kinase and extracellular signal-regulated protein kinases confers cadmium-induced HSP70 expression in 9L rat brain tumor cells

We have reported that treatment with CdCl2 at 40-100 microM induces the heat shock proteins (HSPs) in 9L rat brain tumor cells, during which the activation of heat shock factor (HSF) is essentially involved. By exploiting protein kinase inhibitors, we further analyzed the possible participation of specific protein kinases in the above processes. It was found that induction of HSP70 in cells treated with a high concentration of cadmium (i.e. 100 microM) is preceded by the phosphorylation and activation of p38 mitogen-activated protein kinase (p38(MAPK)), while that in cells treated with a low concentration (60 microM) is accompanied by the phosphorylation and activation of extracellular-regulated protein kinases 1 and 2 (ERK1/2). In 100 microM cadmium-treated cells, both HSP70 induction and HSF1 activation are eliminated in the presence of SB203580, a specific inhibitor of p38(MAPK). By contrast, in 60 microM cadmium-treated cells, the processes are not affected by SB203580 but are significantly suppressed by PD98059, which indirectly inhibits ERK1/2 by acting on MAPK-ERK kinase. Taken together, we demonstrate that p38(MAPK) and ERK1/2 can be simultaneously or independently activated under different concentrations of cadmium and that the signaling pathways participate in the induction of HSP70 by acting on the inducible phosphorylation of HSF1. We thus provide the first evidence that both p38(MAPK) and ERK signaling pathways can differentially participate in the activation of HSF1, which leads to the induction of HSP70 by cadmium.     

Changes in the distribution of intermediate filaments in rat Sertoli cells during the seminiferous epithelium cycle and postnatal development

BACKGROUND: Intermediate filaments (IFs) are components of the cytoskeleton. In mammalian Sertoli cell, IFs are formed by vimentin. Previous studies have shown some characteristics of its distribution in Sertoli cells, however, very little is known of its distributional changes during the seminiferous epithelium cycle and during postnatal development. METHODS: Immunohistochemical and electron microscopic methods were used to determine the distribution of vimentin-type IFs in rat Sertoli cells during the seminiferous epithelium cycle and postnatal development. RESULTS: The distribution of IFs in adult rat Sertoli cell showed distinct cyclic changes during the seminiferous epithelium cycle. At stages I-VI, bundles of IFs extend from the perinuclear region to the supranuclear and apical regions of the Sertoli cell. These apical extensions became shorter at stage VII, and at stages VIII-X IFs were observed only in the perinuclear region. Short apical extensions reappeared at stages XI-XII; and at stages XIII-XIV, they extended again into the apical region. During this cycle, IFs were always closely associated with the heads of elongate spermatids. IFs were also shown to be in close apposition to some specialized structures on the cell membrane, such as the ectoplasmic specialization between adjacent Sertoli cells. During postnatal (p.n.) development, IFs were mainly observed at the basal nuclear region on p.n. day 7. The IFs in the supranuclear or apical regions first appeared at p.n. day 14 and gradually increased during the development. The perinuclear IFs network was fully established by p.n. day 28 and the adult distribution pattern of the IFs was established by p.n. day 42. CONCLUSIONS: Vimentin-type IFs in rat Sertoli cells are a delicate endocellular network, which is centered in the perinuclear region and extends to the apical region of the cell. During the seminiferous epithelium cycle, the distribution of IFs changes in a stage-dependent manner and is closely related to the location of the heads of elongate spermatids. During postnatal development, IFs gradually increase in numbers and the main distribution area is transferred from the basal nuclear to the perinuclear and supranuclear regions.     

Visualization of mitotic radial glial lineage cells in the developing rat brain by Cdc2 kinase-phosphorylated vimentin

It has been reported that cellular oxidative stress induces apoptosis, that may be inhibited by scavengers of reactive oxygen intermediates (ROIs). Superoxide dismutase (SOD) is among the most active scavengers of ROIs, providing defense against the cellular oxidative stress. Fas antigen and tumor necrosis factor (TNF) receptor are the cell surface proteins, stimulation of which induces apoptosis of keratinocytes. Using SV40-transformed human keratinocytes (SVHK cells), we investigated the effects of anti-Fas antibody and TNF-alpha on the SOD activity. Treatment of SVHK cells with anti-Fas antibody or TNF-alpha in the presence of interferon-gamma (IFN-gamma) resulted in an increase in Mn-SOD activity, Cu,Zn-SOD activity was not affected. In the absence of IFN-gamma, no increase in Mn-SOD activity was detected. The induction of IFN-gamma-dependent Mn-SOD activity by anti-Fas antibody or TNF-alpha was concentration-dependent; the maximal effect was observed at 1-10 micrograms/ml and 5-10 ng/ml, respectively. The increase in Mn-SOD activity was observed at 6 h following the treatment and remained for at least 48 h. Northern blot analyses showed that Mn-SOD mRNA increased within 3 h without a significant change in Cu,Zn-SOD mRNA. The addition of both anti-Fas antibody and TNF-alpha in the presence of IFN-gamma resulted in an additive increase in Mn-SOD activity. Although the addition of 12-o-tetradecanoylphorbol-13-acetate (TPA) singly to the incubation medium had no effect on either Mn-, or Cu,Zn-SOD activity, it significantly augmented the IFN-gamma-dependent induction of Mn-SOD activity by anti-Fas antibody or by TNF-alpha. The protein kinase C inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride (H-7), significantly inhibited the TPA-dependent increase in Mn-SOD activity. These results indicate that the stimulation of Fas antigen or TNF receptor increases Mn-SOD activity of SVHK cells in the presence of IFN-gamma and that TPA augments the process through the activation of protein kinase C.     

Evidence for direct binding of intracellularly distributed ganglioside GM2 to isolated vimentin intermediate filaments in normal and Tay-Sachs disease human fibroblasts

The insulinotropic action of meglitinide was compared to that of its analogs S 3075, A-4166, KAD-1229 and repaglinide. None of these hypoglycemic agents significantly enhanced insulin output from rat pancreatic islets incubated for 90 min in the absence of exogenous nutrient. However, all these agents, when tested at a 10 microM concentration, augmented insulin release evoked by either 7 mM D-glucose or 10 microM succinic acid monomethyl ester (SAM). In this respect, meglitinide was a less efficient secretagogue than the other non-sulfonylurea hypoglycemic agents. Moreover, in the presence of 7 mM D-glucose, the lowest concentration of the drug required to cause a significant increase in insulin output decreased from about 1.0 microM for meglitinide to 0.1 microM with A-4166, KAD-1229 or repaglinide and even close to 10 nM in the case of S 3075. The concentration-response relationship thus yielded the following hierarchy, S 3075 > KAD-1229 = repaglinide > A-4166 > meglitinide, there being a difference of more than two orders of magnitude between the weakest and most potent agent.     

Evaluation of lipid-coated microbubbles as a delivery vehicle for Taxol in brain tumor therapy

OBJECTIVE: This work evaluates the potential of lipid-coated microbubbles (LCM) as a delivery vehicle for lipid-soluble antineoplastic agents. We have shown, in rats, the selective affinity of intravenously administered LCM for tumor cells. They are internalized by the tumor cells both in vitro and in vivo. The specificity of LCM for tumors and subsequent incorporation into the cytoplasm could significantly reduce the systemic effects of an agent incorporated into the bubbles, such as Taxol. METHODS: The in vitro methods were as follows. C6 cells (10(5) cells) were treated with Taxol-LCM (6 micrograms/ml), Taxol-Cremophore (6 micrograms/ml), or LCM alone for 8 or 24 hours. Cell death was determined by staining the cells with nuclear staining. Abnormalities of microtubule structures were ascertained by confocal microscopy. The in vivo methods were as follows. Two rat tumor models (C6 and 9L) were used. Rats were treated with single bolus injections or with repetitive (two or three) treatment courses, with respective control animals. Each course consisted of one daily tail vein injection for 5 consecutive days and then 2 days of rest. RESULTS: When compared with either a saline control group or a group receiving Taxol in an oil vehicle, Taxol-LCM reduced tumor progression in Fischer 344 rats inoculated with 9L glioma. The most profound effect was observed with rats treated with three treatment cycles (five daily injections/cycle) separated by two rest periods (2 d/period). CONCLUSION: Both in vitro and in vivo data indicate that Taxol can be incorporated into LCM, can be delivered to the tumor site, and can exert a measurable antitumor biological effect.     

Growth kinetics and treatment response of the intracerebral rat 9L brain tumor model: a quantitative in vivo study using magnetic resonance imaging

We report the use of magnetic resonance imaging (MRI) for in situ tumor growth rate studies of experimental intracranial 9L tumors. T2-weighted spin-echo coronal magnetic resonance images of rat brains with 9L tumors were obtained every 2 days beginning at 8-11 days postimplantation using a 7 tesla MRI system. Tumors were clearly delineated in the images as a hyperintense region with a relatively well-demarcated border and minimal peritumoral edema. Tumor volumes from individual slices were summed together to yield the total tumor volume. The accuracy of this methodology for volumetric determination was verified by MRI phantom studies. Tumor growth rates determined from sequential MRI measurements of tumor volumes were quantitated in terms of volumetric doubling time. Tumor doubling times were found to range from 50 to 81 h, with an average of 66 +/- 8 h (n = 10). Intracranial 9L tumors were found to grow exponentially over the entire life span of the animal, allowing treated animals to serve as their own controls since the volumetric doubling time could be determined from three to four MRI scans before treatment administration. The intracerebral tumor growth delay following a single injection of 1, 3-bis(2-chloroethyl)-1-nitrosourea (13.3 mg/kg i.p.) allowed for noninvasive determination of in vivo log cell kill. A 2.0 +/- 0.2 (n = 3) log cell kill from 1,3-bis(2-chloroethyl)-1-nitrosourea treatment was found from post-treatment MRI volume measurements. These results demonstrate that MRI provides a powerful and sensitive method for assessing the growth and treatment response of intracranial 9L tumors in the rat.     

5-Aminolevulinic acid induced endogenous porphyrin fluorescence in 9L and C6 brain tumours and in the normal rat brain

A new approach in photodynamic therapy is the use of endogenous porphyrins for sensitisation of tumours to light. The induction of endogenous porphyrins after intravenous injection of 5-aminolevulinic acid (ALA, 200 mg kg-1) was studied in 23 rats, bearing intracranial 9L or C6 tumours. After 0, 2, 4, 6, 8, and 22 hours the rats were sacrificed and the fluorescence distribution of endogenous porphyrins was studied in brain tissue sections with a standard fluorescence microscope and a confocal laser scanning microscope. The role of blood-brain barrier disruption on porphyrin production was studied in 2 rats with a cryo-lesion of the cortex. Additionally, 9L and C6 tumour cell cultures were incubated with ALA for 8 hours in vitro. Fluorescence was measured with a fluorescence spectrophotometer in cell cultures and in the brain sections. Porphyrins were detected in vitro in the tumour cells from 2 hours onwards and ex vivo in the tumour sections mainly from 2 to 8 hours, by 22 hours porphyrin fluorescence had almost disappeared. The contralateral brain showed low fluorescence levels between 2 and 6 hours after ALA administration. At the site of the cryo-lesions low fluorescence was measured 6 hours after ALA administration. The 9L tumours fluoresced homogeneously, with a sharp demarcation towards normal brain tissue. Fluorescence in the C6 tumours was patchy, with a poorly fluorescing edge. In both tumour models fluorescence was also detected in brain surrounding the tumour and sometimes in contralateral white matter and ventricle ependyma and pia mater. The slight increase of porphyrin fluorescence in the normal brain of tumour bearing rats, compared to the absence of this in rats without a tumour, was attributed to transport by bulk flow of porphyrins made in the tumours, and possibly also of circulating porphyrins or ALA leaking from the tumour vessels.     

Involvement of heat shock elements and basal transcription elements in the differential induction of the 70-kDa heat shock protein and its cognate by cadmium chloride in 9L rat brain tumor cells

The EphA3 receptor tyrosine kinase has been implicated in guiding the axons of retinal ganglion cells as they extend in the optic tectum. A repulsive mechanism involving opposing gradients of the EphA3 receptor on retinal axons and its ligands, ephrin-A2 and ephrin-A5, in the tectum influences topographic mapping of the retinotectal projection. To investigate the overall role of the Eph family in patterning of the visual system, we have used in situ hybridization to localize nine Eph receptors in the chicken retina and optic tectum at Embryonic Day 8. Three of the receptors examined correspond to the novel chicken homologs of EphA2, EphA6, and EphA7. Unexpectedly, we found that many Eph receptors are expressed not only in retinal ganglion cells, but also in tectal cells, In particular, EphA3 mRNA is prominently expressed in the anterior tectum, with a pattern reciprocal to that of ephrin-A2 and ephrin-A5. Similarly, ephrin-A5 is expressed not only in tectal cells but also in the nasal retina, with a pattern reciprocal to that of its receptor EphA3 and partially overlapping with that of its other receptor EphA4. Consistent with the even distribution of EphA4 and the polarized distribution of EphA4 ligands in the retina, probing EphA4 immunoprecipitates from different sectors of the retina with anti-phosphotyrosine antibodies revealed spatial differences in receptor phosphorylation. These complex patterns of expression and tyrosine phosphorylation suggest that Eph receptors and ephrins contribute to establishing topography of retinal axons through multiple mechanisms, in addition to playing a role in intraretinal and intratectal organization.     

A structural scaffolding of intermediate filaments in health and disease

The cytoplasm of animal cells is structured by a scaffolding composed of actin microfilaments, microtubules, and intermediate filaments. Intermediate filaments, so named because their 10-nanometer diameter is intermediate between that of microfilaments (6 nanometers) and microtubules (23 nanometers), assemble into an anastomosed network within the cytoplasm. In combination with a recently identified class of cross-linking proteins that mediate interactions between intermediate filaments and the other cytoskeletal networks, evidence is reviewed here that intermediate filaments provide a flexible intracellular scaffolding whose function is to structure cytoplasm and to resist stresses externally applied to the cell. Mutations that weaken this structural framework increase the risk of cell rupture and cause a variety of human disorders.     

Expression of intermediate filaments in normal and neoplastic exocrine pancreas

The intermediate filament (IF) proteins present in the normal and pathological exocrine human pancreas were studied by immunolocalization using antibodies to cytokeratins (CKs) and vimentin. Acinar cells of normal pancreas showed a presence of simple CKs 8 and 18. Duct epithelium consistently expressed CKs 7, 8, 18 and 19 whereas centroacinar cells were rather low in CK 7. A subpopulation of CK 4 cells was detected in inter-intralobular ducts. In addition, some ducts contained individual cells or groups of cells that were positive for the stratification-related CKs (CKs 4, 5, 13, 15, 16). All pancreatic ductal adenocarcinomas regularly expressed CKs 7, 8, 18, 19 and were also positive for the 34 beta E12 antibody. Cytokeratin 4 was detected in a minor population of tumor cells. Pancreatic carcinoma also contained minor amounts of stratification-related CKs in variable combinations. Mucinous cystoadenocarcinoma showed the presence of CKs 7, 8, 18, 19 and was also positive for 34 beta E12, whereas the serous microcystic tumor presented CKs 8, 18, 19 and a variable amount of CKs 4 and 7. The duct-ductular alterations of the exocrine pancreas contained a different combination and distribution of CK isoforms similar to normal pancreatic ductal system. Mucinous hypertrophy and pyloric gland metaplasia reacted with antibodies to CKs 7, 8, 18 and 19. Vimentin was focally present both in normal and neoplastic tissue. Our results indicate that pancreatic ducts are characterized by an intrinsic "biliary-pancreatic duct type" immunoprofile (CKs 7, 8, 18 and 19), in contrast to acinar cells expressing exclusively CKs 8 and 18. We also detected a subpopulation of ducts regularly expressing CK 4. Surprisingly, several stratification-related CKs were detected both in normal and neoplastic exocrine pancreas. Moreover, the differentiation phenotypes of pancreatic tumors were reminiscent of normal cellular compartments.