Lan Bo Chen

Dynamic behavior of endoplasmic reticulum in living cells

Endoplasmic reticulum (ER) was studied by fluorescence microscopy of living CV-1 cells treated with the fluorescent carbocyanine dye DiOC6(3). Using video recording and image processing techniques, several distinct forms of highly localized movements of ER were documented, categorized, and analyzed in terms of mechanism and structural implications. These include tubule branching, ring closure, and sliding. These localized movements have been observed to generate the basic elements of ER: linear tubules, polygonal reticulum, and triple junctions. We propose that as such they act as the mechanism for constructing the polygonal lattice of interconnected membrane tubules that constitutes ER. The nature of these movements suggests possible involvement of the cytoskeleton, and, in view of the close correlations in the distributions of ER and microtubules, and the accompanying paper (Dabora and Sheetz), it is possible that microtubules may play a role in generating ER motility and in constructing and maintaining the ER network in living cells.

Localization of endoplasmic reticulum in living and glutaraldehyde-fixed cells with fluorescent dyes

Certain fluorescent dyes, previously reported to localize mitochondria, when used at higher concentrations also localize a continuous net-like structure in both living and glutaraldehyde-fixed cells. A similar reticular structure can be detected by phase-contrast microscopy and whole-mount electron microscopy in potassium permanganate-fixed cells as well. This structure is mostly tubular, with some patch-like areas, and is likely to be the endoplasmic reticulum (ER). The organization of the reticular structure is sensitive to colchicine and rotenone but not to cytochalasin B, taxol, monensin, the calcium ionophore A23187, 12-O-tetradecanoylphorbol 13-acetate, or hydrocortisone.

The intranuclear location of a herpes simplex virus DNA-binding protein is determined by the status of viral DNA replication

The herpes simplex viral DNA-binding protein, ICP8, is targeted to two different locations in the cell nucleus as part of its maturation pathway. Prior to viral DNA synthesis ICP8 was found at discrete pre-replicative sites throughout the nucleus, where it exhibited a high salt-labile association with the nuclear matrix. During viral DNA replication ICP8 was localized in randomly distributed replication compartments, where it is bound to viral DNA. Initiation of viral DNA replication caused the protein to move from the prereplicative sites to the replication compartments, while inhibition of replication caused movement in the opposite direction. In cells where viral DNA synthesis was proceeding, a minor population of ICP8 may also have been associated with the prereplicative sites. The prereplicative sites may serve as a nuclear reservoir for ICP8 not bound to replicating or progeny DNA.

Inactivation of DNA-Dependent Protein Kinase by Protein Kinase Cδ: Implications for Apoptosis

ABSTRACT Protein kinase Cδ (PKCδ) is proteolytically cleaved and activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. A role for PKCδ in apoptosis is supported by the finding that overexpression of the catalytic fragment of PKCδ (PKCδ CF) in cells is associated with the appearance of certain characteristics of apoptosis. However, the functional relationship between PKCδ cleavage and induction of apoptosis is unknown. The present studies demonstrate that PKCδ associates constitutively with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The results show that PKCδ CF phosphorylates DNA-PKcs in vitro. Interaction of DNA-PKcs with PKCδ CF inhibits the function of DNA-PKcs to form complexes with DNA and to phosphorylate its downstream target, p53. The results also demonstrate that cells deficient in DNA-PK are resistant to apoptosis induced by overexpressing PKCδ CF. These findings support the hypothesis that functional interactions between PKCδ and DNA-PK contribute to DNA damage-induced apoptosis.

BCR/ABL induces multiple abnormalities of cytoskeletal function.

The BCR/ABL oncogene causes human chronic myelogenous leukemia (CML), a myeloproliferative disease characterized by massive expansion of hematopoietic progenitor cells and cells of the granulocyte lineage. When transfected into murine hematopoietic cell lines, BCR/ABL causes cytokine-independence and enhances viability. There is also growing evidence that p210(BCR/ABL) affects cytoskeletal structure. p210(BCR/ABL) binds to actin, and several cytoskeletal proteins are tyrosine phosphorylated by this oncoprotein. Also, at least one aspect of cytoskeletal function is abnormal, in that the affinity of beta1 integrins for fibronectin is altered in CML cells. However, isolated changes in beta1 integrin function would be unlikely to explain the clinical phenotype of CML. We used time-lapse video microscopy to study cell motility and cell morphology on extracellular cell matrix protein-coated surfaces of a series of cell lines before and after transformation by BCR/ABL. BCR/ABL was associated with a striking increase in spontaneous motility, membrane ruffling, formation of long actin extensions (filopodia) and accelerated the rate of protrusion and retraction of pseudopodia on fibronectin-coated surfaces. Also, while untransformed cells were sessile for long periods, BCR/ABL-transformed cells exhibited persistent motility, except for brief periods during cell division. Using cell lines transformed by a temperature-sensitive mutant of BCR/ABL, these kinetic abnormalities of cytoskeletal function were shown to require BCR/ABL tyrosine kinase activity. Similar abnormalities of cytoskeletal function on fibronectin-coated surfaces were observed when hematopoietic progenitor cells purified by CD34 selection from patients with CML were compared with CD34 positive cells from normal individuals. Interestingly, alpha-interferon treatment was found to slowly revert the abnormal motility phenotype of BCR/ABL-transformed cells towards normal. The increase in spontaneous motility and other defects of cytoskeletal function described here will be useful biological markers of the functional effects of BCR/ABL in hematopoietic cells.

Identification of genes regulated by dexamethasone in multiple myeloma cells using oligonucleotide arrays.

Our previous studies have characterized Dexamethasone (Dex)-induced apoptotic signaling pathways in multiple myeloma (MM) cells; however, related transcriptional events are not fully defined. In the present study, gene expression profiles of Dex-treated MM cells were determined using oligonucleotide arrays. Dex triggers early transient induction of many genes involved in cell defense/repair-machinery. This is followed by induction of genes known to mediate cell death and repression of growth/survival-related genes. The molecular and genetic alterations associated with Dex resistance in MM cells are also unknown. We compared the gene expression profiles of Dex-sensitive and Dex-resistant MM cells and identified a number of genes which may confer Dex-resistance. Finally, gene profiling of freshly isolated MM patient cells validates our in vitro MM cell line data, confirming an in vivo relevance of these studies. Collectively, these findings provide insights into the basic mechanisms of Dex activity against MM, as well as mechanisms of Dex-resistance in MM cells. These studies may therefore allow improved therapeutic uses of Dex, based upon targeting genes that regulate MM cell growth and survival.

Multiple Molecular Chaperones Complex with Misfolded Large Oligomeric Glycoproteins in the Endoplasmic Reticulum

Thyroglobulin (Tg), the major protein secreted by thyroid epithelial cells and precursor of thyroid hormones, is a large dimeric glycoprotein with multiple disulfide bonds. The folding and assembly of this complex molecule begins in the endoplasmic reticulum (ER) and is likely to involve a variety of reactions catalyzed by molecular chaperones (Kuznetsov, G., Chen, L. B., and Nigam, S. K. (1994) J. Biol. Chem. 269, 22990-22995). By coimmunoprecipitation in rat thyroid cells, we were able to demonstrate that BiP, grp94, ERp72, and grp170, four proteins believed to function as specific molecular chaperones, complex with Tg during its maturation. The same complex of the four putative chaperones with Tg was observed in cells treated with tunicamycin, indicating that these four ER chaperones stably associate with Tg when it is misfolded/misassembled due to inhibition of its glycosylation. BiP, grp94, and ERp72 were also found to associate with Tg in cells in which misfolding was induced by perturbing ER calcium stores. To determine if the assembly of a complex between the four chaperones and Tg under conditions of misglycosylation was unique to the maturation of this particular secretory protein or a more general phenomenon, adenovirus-transformed rat thyroid cells that do not synthesize Tg were analyzed. In these transformed cells, the only protein these same four chaperones were found to complex with was a protein of approximately 200 kDa. This protein was subsequently identified as thrombospondin, which, like Tg, is a large oligomeric secreted glycoprotein with multiple disulfide bonds. We therefore propose that these ER chaperones complex together with a variety of large oligomeric secretory glycoproteins as they fold and assemble in the ER.

Elevated serum periostin levels in patients with bone metastases from breast but not lung cancer.

Periostin is a recently identified gene that is preferentially expressed in periosteum, indicating a potential role in bone formation and maintenance of structure. We independently identified and isolated periostin from cancer tissue, using the palindromic PCR-driven cDNA Differential Display technique. For the present work, we developed a novel sandwich chemiluminescence assay to detect serum periostin level using newly developed monoclonal and polyclonal antibodies. We investigated serum periostin levels in breast cancer and small cell lung cancer patients, especially in patients with bone metastasis. The study included 58 breast cancer and 44 small cell lung cancer patients. Serum periostin levels were elevated in breast cancer patients presenting with bone metastases (92.0 ± 28.6 ng/ml) compared to similar breast cancer patients without evidence of bone metastasis (55.0 ± 16.6 ng/ml, p = 0.04). No correlation was found between the serum periostin level and any other prognostic factors, such as clinical stage and lymph node metastasis in breast cancer. Serum periostin levels thus appear to serve as a marker of bone metastasis from breast cancer. In contrast, serum periostin levels were similar in samples from patients with small cell lung cancer who did or did not have bone metastasis. However, increasing T-stage and N-stage of patients with small cell lung cancer were correlated with higher periostin levels (T4, 126.5 ± 29.7 ng/ml v.s. T2, 64.9 ± 16.1 ng/ml, p = 0.03; and T4 v.s. T1, 36.3 ± 7.5 ng/ml, p = 0.01; N3, 108.7 ± 17.3 ng/ml v.s. N2, 49.7 ± 10.9 ng/ml, p = 0.01). Periostin has a substantial homology with the insect cell adhesion molecule, fasciclin I. Thus, expression of periostin may facilitate tumor cell adhesion to the bone surface. In fact, we found by in situ RNA hybridization, that the periostin gene was highly expressed in the stromal cells immediately surrounding the tumor, but not within the breast cancer cells themselves.

Rhodamine 123 inhibits bioenergetic function in isolated rat liver mitochondria

Rhodamine 123 accumulates in the mitochondria of living cells and exhibits selective anticarcinoma activity. The biochemical basis of toxicity was investigated by testing the effect of the dye on isolated rat liver mitochondria. Much lower concentrations of rhodamine 123 were required to inhibit ADP-stimulated respiration and ATP synthesis in well-coupled energized mitochondria than were required to inhibit uncoupled respiration and uncoupler-stimulated ATP hydrolysis. The amount of rhodamine 123 associated with the mitochondria was several-fold greater under energized as compared to non-energized conditions, which may explain why coupled functions appeared to be more sensitive than uncoupled functions to inhibition at low concentrations of rhodamine 123. It was concluded that the site of rhodamine 123 inhibition is most likely the F0F1 ATPase complex and possibly electron transfer reactions as well.

p130CAS Forms a Signaling Complex with the Adapter Protein CRKL in Hematopoietic Cells Transformed by the BCR/ABL Oncogene

The Philadelphia chromosome (Ph) translocation generates a chimeric tyrosine kinase oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML) and a type of acute lymphoblastic leukemia (ALL). In primary samples from virtually all patients with CML or Ph+ALL, the CRKL adapter protein is tyrosine phosphorylated and physically associated with p210BCR/ABL. CRKL has one SH2 domain and two SH3 domains and is structurally related to c-CRK-II (CRK) and the v-Crk oncoprotein. We have previously shown that CRKL, but not the related adapter protein c-CRK, is tyrosine phosphorylated in cell lines transformed by BCR/ABL, and that CRKL binds to BCR/ABL through the CRKL-SH3 domains. Furthermore, the CRKL-SH2 domain has been shown to bind one or more cellular proteins, one of which is p120CBL. Here we demonstrate that another cellular protein linked to BCR/ABL through the CRKL-SH2 domain is p130CAS. p130CAS was found to be tyrosine phosphorylated and associated with CRKL in BCR/ABL expressing cell lines and in samples obtained from CML and ALL patients, but not in samples from controls. In both normal and BCR/ABL transformed cells, p130CAS was detected in focal adhesion-like structures, as was BCR/ABL. In normal cells, the focal adhesion proteins tensin, p125FAK, and paxillin constitutively associated with p130CAS. However, in BCR/ABL transformed cells, the interaction between p130CAS and tensin was disrupted, while the associations between p130CAS, p125FAK, and paxillin were unaffected. These results suggest that the BCR/ABL oncogene could alter the function of p130CAS in at least three ways: tyrosine phosphorylation, inducing constitutive binding of CRKL to a domain in p130CAS containing Tyr-X-X-Pro motifs (substrate domain), and disrupting the normal interaction of p130CAS with the focal adhesion protein tensin. These alterations in the structure of signaling proteins in focal adhesion like structures could contribute to the known adhesion abnormalities in CML cells.