Electron microscopy evidence that cytoplasmic localization of the p16INK4A "nuclear" cyclin-dependent kinase inhibitor (CKI) in tumor cells is specific and not an artifact. A study in non-small cell lung carcinomas

It is well established that p16^INK4A protein acts as a cell cycle inhibitor in the nucleus. Therefore, cytoplasmic localization of p16 ^INK4A usually is disregarded by investigators as nonspecific. Three recent studies reported findings that differ from the current view concerning p16^INK4A immunohistochemical localization. All three demonstrated that breast and colon cancers expressing cytoplasmic p16^INK4 represent distinct biological subsets. We previously detected in a percentage of non-small cell lung carcinomas simultaneous nuclear and cytoplasmic p16^INK4A staining. In view of the reports concerning breast and colon carcinomas, we conducted an ultrastructural re-evaluation of our cases to clarify the specificity of p16^INK4A cytoplasmic expression. We observed p16 ^INK4A immunolocalization in both the nucleus and the cytoplasm of a proportion of tumor cells. Diffuse dense nuclear staining was detected in the nucleoplasm, whereas weaker granular immunoreactivity was observed in the cytoplasm near the rough endoplasmic reticulum. Negative tumor cells also were visible. In the tumor-associated stromal, cells p16^INK4A immunoreactivity was detected only in the nuclei. We have demonstrated that p16^INK4A cytoplasmic staining is specific and suggest that it represents a mechanism of p16^INK4A inactivation similar to that observed in other tumor suppressor genes.     

Electron microscopy evidence that cytoplasmic localization of the p16INK4A “nuclear” cyclin-dependent kinase inhibitor (CKI) in tumor cells is specific and not an artifact. A study in non-small cell lung carcinomas

It is well established that p16^INK4A protein acts as a cell cycle inhibitor in the nucleus. Therefore, cytoplasmic localization of p16 ^INK4A usually is disregarded by investigators as nonspecific. Three recent studies reported findings that differ from the current view concerning p16^INK4A immunohistochemical localization. All three demonstrated that breast and colon cancers expressing cytoplasmic p16^INK4 represent distinct biological subsets. We previously detected in a percentage of non-small cell lung carcinomas simultaneous nuclear and cytoplasmic p16^INK4A staining. In view of the reports concerning breast and colon carcinomas, we conducted an ultrastructural re-evaluation of our cases to clarify the specificity of p16^INK4A cytoplasmic expression. We observed p16 ^INK4A immunolocalization in both the nucleus and the cytoplasm of a proportion of tumor cells. Diffuse dense nuclear staining was detected in the nucleoplasm, whereas weaker granular immunoreactivity was observed in the cytoplasm near the rough endoplasmic reticulum. Negative tumor cells also were visible. In the tumor-associated stromal, cells p16^INK4A immunoreactivity was detected only in the nuclei. We have demonstrated that p16^INK4A cytoplasmic staining is specific and suggest that it represents a mechanism of p16^INK4A inactivation similar to that observed in other tumor suppressor genes.     

Tissue and subcellular distribution of glucokinase in rat liver and their changes during fasting-refeeding

The distribution of glucokinase in rat liver under both normal feeding and fasting-refeeding conditions was investigated immunohistochemically. Under normal feeding conditions, glucokinase immunoreactivity was observed in both nuclei and cytoplasm of parenchymal cells. The nuclei were stained intensely and evenly, whereas the cytoplasm showed weak immunoreactivity of different degrees of staining intensity depending on the location of the cells. The cytoplasm of perivenous hepatocytes was stained more intensely, though not so much more, than that of periportal hepatocytes. The cytoplasm of hepatocytes surrounding the terminal hepatic venule (THV), of hepatocytes surrounding the portal triad, and of some other hepatocytes showed a stronger immunoreactivity than that of residual hepatocytes. The nuclear immunoreactivity in hepatocytes surrounding the portal triad and in some other hepatocytes was weak or absent, and positive immunoreactivity was detected at the plasma membrane of some of these cells. After 72 h of fasting, glucokinase immunoreactivity was markedly decreased in all hepatocytes. After the start of refeeding, the cytoplasmic immunoreactivity began to increase first in the parenchymal cells surrounding the THV and extended to those in the intermediate zone followed by those in the periportal zone. In contrast, the increase in nuclear immunoreactivity started in hepatocytes situated in the intermediate zone adjacent to the perivenous zone and then extended to those in the perivenous zone followed by those in the periportal zone. Hepatocytes surrounding either THV or portal triad showed a distinctive change in immunoreactivity during the refeeding period. After 10 h of refeeding, strong immunoreactivity was observed in both the cytoplasm and the nuclei of all hepatocytes, and appreciable glucokinase immunoreactivity was detected at the plasma membrane of some hepatocytes. These findings are discussed from the standpoint of a functional role of glucokinase in hepatic glucose metabolism.     

Cytoplasmic retention of protein tyrosine kinase 6 promotes growth of prostate tumor cells

Protein tyrosine kinase 6 (PTK6) is an intracellular tyrosine kinase that is nuclear in epithelial cells of the normal prostate, but cytoplasmic in prostate tumors and in the PC3 prostate tumor cell line. The impact of altered PTK6 intracellular localization in prostate tumor cells has not been extensively explored. Knockdown of endogenous cytoplasmic PTK6 resulted in decreased PC3 cell proliferation and colony formation, suggesting that cytoplasmic PTK6 stimulates oncogenic pathways. In contrast, reintroduction of PTK6 into nuclei of PC3 cells had a negative effect on growth. Enhanced tyrosine phosphorylation of the PTK6 substrate Sam68 was detected in cells expressing nuclear-targeted PTK6. We found that mechanisms regulating nuclear localization of PTK6 are intact in PC3 cells. Transiently over-expressed PTK6 readily enters the nucleus. Ectopic expression of ALT-PTK6, a catalytically inactive splice variant of PTK6, did not affect localization of endogenous PTK6 in PC3 cells. Using leptomycin B, we confirmed that cytoplasmic localization of endogenous PTK6 is not due to CRM-1/exportin-1 mediated nuclear export. In addition, over-expression of the PTK6 nuclear substrate Sam68 is not sufficient to bring PTK6 into the nucleus. While exogenous PTK6 was readily detected in the nucleus when transiently expressed at high levels, low-level expression of inducible wild type PTK6 in stable cell lines resulted in its cytoplasmic retention. Our results suggest that retention of PTK6 in the cytoplasm of prostate cancer cells disrupts its ability to regulate nuclear substrates and leads to aberrant growth. In prostate cancer, restoring PTK6 nuclear localization may have therapeutic advantages.     

Cytoplasmic retention of protein tyrosine kinase 6 promotes growth of prostate tumor cells

Protein tyrosine kinase 6 (PTK6) is an intracellular tyrosine kinase that is nuclear in epithelial cells of the normal prostate, but cytoplasmic in prostate tumors and in the PC3 prostate tumor cell line. The impact of altered PTK6 intracellular localization in prostate tumor cells has not been extensively explored. Knockdown of endogenous cytoplasmic PTK6 resulted in decreased PC3 cell proliferation and colony formation, suggesting that cytoplasmic PTK6 stimulates oncogenic pathways. In contrast, reintroduction of PTK6 into nuclei of PC3 cells had a negative effect on growth. Enhanced tyrosine phosphorylation of the PTK6 substrate Sam68 was detected in cells expressing nuclear-targeted PTK6. We found that mechanisms regulating nuclear localization of PTK6 are intact in PC3 cells. Transiently overexpressed PTK6 readily enters the nucleus. Ectopic expression of ALT-PTK6, a catalytically inactive splice variant of PTK6, did not affect localization of endogenous PTK6 in PC3 cells. Using leptomycin B, we confirmed that cytoplasmic localization of endogenous PTK6 is not due to Crm-1/exportin-1 mediated nuclear export. In addition, overexpression of the PTK6 nuclear substrate Sam68 is not sufficient to bring PTK6 into the nucleus. While exogenous PTK6 was readily detected in the nucleus when transiently expressed at high levels, low-level expression of inducible wild type PTK6 in stable cell lines resulted in its cytoplasmic retention. Our results suggest that retention of PTK6 in the cytoplasm of prostate cancer cells disrupts its ability to regulate nuclear substrates and leads to aberrant growth. In prostate cancer, restoring PTK6 nuclear localization may have therapeutic advantages.Key words: PTK6, BRK, Sik, SAM68, prostate cancer, nuclear localization, tyrosine kinase     

Light-dependent PER-like proteins in the cephalic ganglia of an apterygote and a pterygote insect species

Antibodies targeted to a highly conserved tetradecapeptide region of the pivotal biological clock protein PER detect in the firebrat Thermobia domestica a 115-kDa protein and in the cockroach Periplaneta americana a 110-kDa protein that are present in the cytoplasm of a small set of brain cells. A similar cytoplasmic reaction occurs with antisera to the whole PER protein of Drosophila melanogaster, but these antisera also react with numerous cell nuclei. On western blots, they detect an 80-kDa antigen in T. domestica and 70- and 80-kDa antigens in P. americana. No indication of antigen translocation between cell nuclei and cytoplasm was found. Nuclear staining is maintained at a high constant level in T. domestica held at a 12:12 h light:dark photoperiod (LD) or in continuous light, but disappears rapidly in response to extended darkness. In P. americana under LD conditions, the number of immunoreactive nuclei and their staining intensity fluctuate in parallel, with maximal staining late in the day. The circadian changes are maintained in continuous light but all staining vanishes in continuous darkness. A 6-h light pulse in early night of an LD cycle induces maximal staining after about 10 h, suggesting that the effect of light on nuclear PER-like expression is indirect. The behaviour of nuclear antigens is opposite to that of the cytoplasmic PER-like proteins that persist in constant darkness and disappear in constant light. Under LD conditions, the cytoplasmic PER-like antigen cycles in T. domestica but remains at a steady level in P. americana. The sensitivity to photoregime suggests that both the nuclear and the cytoplasmic PER-like antigens are components of the biological clock.R. Závodská and H. Sehadová contributed equally to this work     

The myc proteins are not associated with chromatin in mitotic cells.

The protein products of cellular and viral myc oncogenes are detected in nuclei by immunofluorescence. No myc fluorescence is found in nucleoli. In mitotic cells the myc antigens are not found associated with metaphase chromosomes, but are diffusely distributed throughout the cytoplasm. Cytoplasmic myc fluorescence is first observed when chromatin begins to condense in early prophase. Granular nuclear myc fluorescence is again discerned in telophase cells, when the nuclear envelope is formed and becomes more prominent upon cytokinesis; concomitantly the diffuse cytoplasmic myc staining is lost. These results suggest that myc proteins not only bind to DNA or chromatin, but are also associated with other structural systems in the nuclei.     

Immunocytochemical localization of glucocorticoid receptors in cells, cytoplasts, and nucleoplasts

A monoclonal antibody has been used to assess the intracellular localization of the glucocorticoid receptor in rodent L-929 fibroblasts and GH3 pituitary tumor cells. Whole cells from both cell lines showed immunoreactivity in the cytoplasm and nucleus. However, when cytoplasts and nucleoplasts of these cells were examined, only L-cells showed strong antibody binding in both fractions; in contrast, GH3 cells exhibited nuclear staining and slight cytoplasmic staining. These results are discussed in terms of the current findings regarding the intracellular location of steroid hormone receptors.     

Spectrin localization in osteoclasts: Immunocytochemistry,cloning, and partial sequencing

The presence of spectrin was demonstrated in chick osteoclasts by Western blotting and light and electron microscopic immunolocalization. Additionally, screening of a chick osteoclast cDNA library revealed the presence of α-spectrin. Light microscope level immunocytochemical staining of osteoclasts in situ revealed spectrin staining throughout the cytoplasm with heavier staining found at the marrow-facing cell margin and around the nuclei. Confocal microscopy of isolated osteoclasts plated onto a glass substrate showed that spectrin encircled the organelle-rich cell center. Nuclei and cytoplasmic inclusions were also stained and the plasma membrane was stained in a nonuniform, patchy distribution corresponding to regions of apparent membrane ruffling. Ultracytochemical localization showed spectrin to be found at the plasma membrane and distributed throughout the cytoplasm with especially intense staining of the nuclear membrane and filaments within the nuclear compartment. J. Cell. Biochem. 71:204–215, 1998. © 1998 Wiley-Liss, Inc.     

Localization of Basic Fibroblast Growth Factor in Bovine Endothelial Cells: Immunohistochemical and Biochemical Studies

To gain insight into the mechanisms of synthesis, storage, and release of basic fibroblast growth factor (bFGF), we studied the immunohistochemical localization of bFGF in bovine coronary artery, coronary sinus, and adrenal capillary endothelial cells grown in culture. Light and electron microscopic immunohistochemical studies were performed using the ABC immunoperoxidase method on p-formaldehyde-fixed cells. Five different anti-bFGF antibodies gave similar results in all cell types. In subconfluent cells, immunoreactivity was noted in the nuclear chromatin, nucleoli, cytosol, cytoplasmic vesicles (some of which appeared to fuse with the plasma membrane), and extracellular matrix. No reaction was found in endoplasmic reticulum or Golgi zones. Confluent cells demonstrated less immunoreactivity in the nuclei and cytosol but more in the extracellular matrix. Some cells of senescent morphology showed only cytoplasmic staining; however, no cells were found with only nuclear staining. Biochemical studies showed that three forms of bFGF (18, 24, and 26 kDa) were present in endothelial cells and varied with different culture conditions. Protection analysis indicated that bFGF mRNA is less abundant in postconfluent cells than in subconfluent cells. These data suggest that subconfluent cells synthesize bFGF and transport it into the nucleus and exocytotic vesicles, while confluent cells synthesize little bFGF but store it in extracellular matrix, cytoplasmic vesicles, and nuclei.     

Lectin receptor sites on rat liver cell nuclear membranes.

The presence and localization of lectin receptor sites on rat liver cell nuclear and other endomembranes was studied by light and electron microscopy using fluorescein and ferritin-coupled lectin conjugates. Isolated nuclei labelled with fluorescein-conjugated Concanavalin A (Con A) or wheat germ agglutinin (WGA) often showed membrane staining, which sometimes was especially bright on small stretches of the nuclear surface. Unlabelled nuclei and nuclei with a complete ring fluorescence were also seen. The nuclear fluorescence corresponded in intensity to that seen on the surface of isolated rat liver cells. Con A-ferritin particles were seldom detected on the cytoplasmic surface of the intact nuclear envelope. However, at places where the 2 leaflets of the envelope were widely separated or where the outer nuclear membrane was partly torn away, heavy labelling was seen on the cisternal surface of both the inner and outer nuclear membranes. Labelling with Con A-ferritin was also found on the cisternal side of rough endoplasmic reticulum present in the specimens. No labelling was seen on the cytoplasmic surface of mitochondrial outer membrane. The results demonstrate the presence of binding sites for Con A and WGA in nuclei and an asymmetric localization of these sites on the cisternal side of ribosome-carrying endomembranes in rat liver cells.     

Direct evidence for nuclear and cytoplasmic colocalization of proteasomes (multiprotease complexes) in liver

Subcellular localization of the large multicatalytic protease complexes called proteasomes, which have been found in soluble fractions of various cells, was examined by biochemical, immunological, and immunohistological methods. Rat liver nuclei, purified by two different procedures, showed high activities for degrading [3H]methylcasein and various fluorogenic oligopeptides with neutral and weakly alkaline pH optima. On gel filtration, all of these peptidase activities were recovered in a single peak with the unusually large molecular weight of about 600,000. Properties of the proteolytic activity in crude extracts of the nucleus and the cytoplasm were very similar. Immunoelectrophoretic blot analysis showed the presence of appreciable concentrations of proteasomes with similar immunoreactivity in isolated nuclear and cytosolic fractions. Moreover, immunohistochemical staining of human liver showed that proteasomes were predominantly localized in the nuclear matrix but also were present diffusely in the cytoplasm of hepatocytes. These findings indicate the nuclear and cytoplasmic colocalization of proteasomes.     

c-fos和c-myc在北方山溪鲵精子发生中的表达

用免疫组织化学方法检测原癌基因cf-os和c-myc蛋白在北方山溪鲵(Batrachuperus tibetanus)精子发生中的表达定位。结果显示,在精原细胞缓慢增殖期,8、9月,FOS阳性反应物出现在精原细胞的胞质及核膜外,10、11月,FOS在少量精原细胞的胞核中表达。在精原细胞快速增殖期,即翌年4月,FOS定位在精原细胞的胞质中;5月,FOS在大量的胞核中强阳性表达;6月,FOS定位于部分精母细胞核质和核膜下;7月,FOS在一些精子细胞的核质和核膜下表达。MYC在8、9月的部分精原细胞胞质中表达较弱,在101、1月阳性反应出现在个别精原细胞的核质中。翌年4月,MYC在精原细胞核周围的胞质中表达;5月在大量的精原细胞核膜下有强表达;6月,MYC在一些精母细胞核膜下表达;7月,MYC在部分精子细胞的核膜下弱表达。结果表明,北方山溪鲵的原癌基因cf-os和c-myc表达大强度在生精细胞发育中呈阶段性,表达的强度和细胞数量与细胞增殖的速度相一致。FOS和MYC在精原细胞内从胞质向胞核的转移与细胞快速增殖的时期相吻合。说明cf-os和c-myc对精原细胞有丝分裂有促进作用,并参与精母细胞成熟分裂的调控。     

Bone microenvironment and androgen status modulate subcellular localization of ErbB3 in prostate cancer cells

ErbB-3, an ErbB receptor tyrosine kinase, has been implicated in the pathogenesis of several malignancies, including prostate cancer. We found that ErbB-3 expression was up-regulated in prostate cancer cells within lymph node and bone metastases. Despite being a plasma membrane protein, ErbB-3 was also detected in the nuclei of the prostate cancer cells in the metastatic specimens. Because most metastatic specimens were from men who had undergone androgen ablation, we examined the primary tumors from patients who have undergone hormone deprivation therapy and found that a significant fraction of these specimens showed nuclear localization of ErbB3. We thus assessed the effect of androgens and the bone microenvironment on the nuclear translocation of ErbB-3 by using xenograft tumor models generated from bone-derived prostate cancer cell lines, MDA PCa 2b, and PC-3. In subcutaneous tumors, ErbB-3 was predominantly in the membrane/cytoplasm; however, it was present in the nuclei of the tumor cells in the femur. Castration of mice bearing subcutaneous MDA PCa 2b tumors induced a transient nuclear translocation of ErbB-3, with relocalization to the membrane/cytoplasm upon tumor recurrence. These findings suggest that the bone microenvironment and androgen status influence the subcellular localization of ErbB-3 in prostate cancer cells. We speculate that nuclear localization of ErbB-3 may aid prostate cancer cell survival during androgen ablation and progression of prostate cancer in bone.     

Glypican and Biglycan in the Nuclei of Neurons and Glioma Cells: Presence of Functional Nuclear Localization Signals and Dynamic Changes in Glypican During the Cell Cycle

We have investigated the expression patterns and subcellular localization in nervous tissue of glypican, a major glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan that is predominantly synthesized by neurons, and of biglycan, a small, leucine-rich chondroitin sulfate proteoglycan. By laser scanning confocal microscopy of rat central nervous tissue and C6 glioma cells, we found that a significant portion of the glypican and biglycan immunoreactivity colocalized with nuclear staining by propidium iodide and was also seen in isolated nuclei. In certain regions, staining was selective, insofar as glypican and biglycan immunoreactivity in the nucleus was seen predominantly in a subpopulation of large spinal cord neurons. The amino acid sequences of both proteoglycans contain potential nuclear localization signals, and these were demonstrated to be functional based on their ability to target β-galactosidase fusion proteins to the nuclei of transfected 293 cells. Nuclear localization of glypican β-galactosidase or Fc fusion proteins in transfected 293 cells and C6 glioma cells was greatly reduced or abolished after mutation of the basic amino acids or deletion of the sequence containing the nuclear localization signal, and no nuclear staining was seen in the case of heparan sulfate and chondroitin sulfate proteoglycans that do not possess a nuclear localization signal, such as syndecan-3 or decorin (which is closely related in structure to biglycan). Transfection of COS-1 cells with an epitope-tagged glypican cDNA demonstrated transport of the full-length proteoglycan to the nucleus, and there are also dynamic changes in the pattern of glypican immunoreactivity in the nucleus of C6 cells both during cell division and correlated with different phases of the cell cycle. Our data therefore suggest that in certain cells and central nervous system regions, glypican and biglycan may be involved in the regulation of cell division and survival by directly participating in nuclear processes.     

Nuclear and nucleolar localization of the 72,000-dalton heat shock protein in heat-shocked mammalian cells

The intracellular location of the major induced mammalian heat shock (or stress) protein (Mr = 72,000) has been determined by both biochemical and immunological methods. This protein, shown here to be comprised of at least three structurally related isoforms, is produced at high levels within 30 min to 1 h following heat treatment of cells. Biochemical fractionation of cells grown under heat shock showed that following its synthesis a portion of the 72,000-Da protein (and its isoforms) becomes associated with the nucleus while some remains in the cytoplasm. Indirect immunofluorescence studies using antiserum directed against the major isoforms of the 72,000-Da protein were carried out in normal and heat-shocked cells as well as in cells grown under stress by exposure to either an amino acid analogue or to sodium arsenite. Diffuse cytoplasmic and nuclear staining was observed in cells grown at 37 degrees C. In cells grown under heat shock conditions, both the cytoplasmic staining and the nuclear staining were found to increase with the nuclear staining consisting of both granular and patch-like structures, the latter being coincident with phase-dense nucleoli. In the case of cells exposed to amino acid analogues or to sodium arsenite, only cytoplasmic and to a lesser extent nuclear staining was observed, i.e. no localized nucleolar fluorescence was observed. Following return of heat shock-treated cells to normal growth temperatures, both the synthesis of the 72,000-Dalton stress protein and its nucleolar staining were found to diminish.     

Subcellular distribution of DNA-binding proteins from cultured hamster fibroblasts

The distribution between nuclei and cytoplasm of DNA-binding proteins from growing NIL cells was studied. To obtain the subcellular fractions, cell monolayers or cells previously detached from the culture dish were treated with the non-ionic detergent Nonidet P-40. Proteins with affinity for DNA were isolated from nuclear or cytoplasmic fractions by chromatography on DNA-cellulose columns and were further analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results show that P8, one of the major components in the 0.15 M NaCl-eluted proteins, is found predominantly in the cytoplasmic fractions, whereas P6, the other main protein peak in this eluate, is more prominent in the nuclear fraction. Among the other proteins eluted at 0.15 M NaCl from the DNA-cellulose column, P5 and P5′ are detected in both nuclear and cytoplasmic fractions. All the other proteins in the 0.15 M NaCl eluate are present almost exclusively in the cytoplasmic fraction. On the other hand, most of the proteins with higher affinity for DNA, eluted from the column at 2 M NaCl, are present in the nuclear fraction, although they are also detected in the cytoplasm in amounts similar to those observed in the nuclei.     

Galectin-3 expression and subcellular localization in senescent human fibroblasts

Galectin-3 is a galactose/lactose-binding protein (M(r) approximately 30,000), identified as a required factor in the splicing of pre-mRNA. In the LG1 strain of human diploid fibroblasts, galectin-3 could be found in both the nucleus and the cytoplasm of young, proliferating cells. In contrast, the protein was predominantly cytoplasmic in senescent LG1 cells that have lost replicative competence through in vitro culture. Incubation of young cells with leptomycin B, a drug that disrupts the interaction between the leucine-rich nuclear export signal and its receptor, resulted in the accumulation of galectin-3 inside the nucleus. In senescent cells, galectin-3 staining remained cytoplasmic even in the presence of the drug, thus suggesting that the observed localization in the cytoplasm was due to a lack of nuclear import. In heterodikaryons derived from fusion of young and senescent LG1 cells, the predominant phenotype was galectin-3 in both nuclei. These results suggest that senescent LG1 cells might lack a factor(s) specifically required for galectin-3 nuclear import.