Inhibition of triple-negative and Herceptin-resistant breast cancer cell proliferation and migration by Annexin A2 antibodies.

BACKGROUND: Annexin A2 (AnxA2), a calcium-dependent phospholipid binding protein, is abundantly present at the surface of triple-negative and Herceptin-resistant breast cancer cells. Interactions between cell-surface AnxA2 and tyrosine kinase receptors have an important role in the tumour microenvironment and act together to enhance tumour growth. The mechanism supporting this role is still unknown. METHODS: The membrane function of AnxA2 was blocked by incubating cells with anti-AnxA2 antibodies. Western blotting, immunoprecipitation, immunofluorescence, 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), flow cytometry, Clonogenic, and wound-healing assays were performed in this study. RESULTS: We demonstrate that AnxA2 interacts with epidermal growth factor receptor (EGFR) at the cell surface and has an important role in cancer cell proliferation and migration by modulating EGFR functions. Blocking AnxA2 function at the cell surface by anti-AnxA2 antibody suppressed the EGF-induced EGFR tyrosine phosphorylation and internalisation by blocking its homodimerisation. Furthermore, addition of AnxA2 antibody significantly inhibited the EGFR-dependent PI3K-AKT and Raf-MEK-ERK downstream pathways under both EGF-induced and basal growth conditions, resulting in lower cell proliferation and migration. CONCLUSIONS: These findings suggest that cell-surface AnxA2 has an important regulatory role in EGFR-mediated oncogenic processes by keeping EGFR signalling events in an activated state. Therefore, AnxA2 could potentially be used as a therapeutic target in triple-negative and Herceptin-resistant breast cancers.

Surface functionalization of PLGA nanoparticles by non-covalent insertion of a homo-bifunctional spacer for active targeting in cancer therapy.

This work reports the surface functionalization of polymeric PLGA nanoparticles by non-covalent insertion of a homo-bifunctional chemical crosslinker, bis(sulfosuccinimidyl) suberate (BS3) for targeted cancer therapy. We dissolved BS3 in aqueous solution of PVA during formulation of nanoparticles by a modified solid/oil/water emulsion solvent evaporation method. The non-covalent insertion of BS3 was confirmed by Fourier transform infrared (FTIR) spectroscopy. Curcumin and annexin A2 were used as a model drug and a cell specific target, respectively. Nanoparticles were characterized for particle size, zeta potential and surface morphology. The qualitative assessment of antibody attachment was performed by transmission electron microscopy (TEM) as well as confocal microscopy. The optimized formulation showed antibody attachment of 86%. However, antibody attachment was abolished upon blocking the functional groups of BS3. The availability of functional antibodies was evaluated by the presence of a light chain fraction after gel electrophoresis. We further evaluated the in vitro release kinetics of curcumin from antibody coated and uncoated nanoparticles. The release of curcumin is enhanced upon antibody attachment and followed an anomalous release pattern. We also observed that the cellular uptake of nanoparticles was significantly higher in annexin A2 positive cells than in negative cells. Therefore, these results demonstrate the potential use of this method for functionalization as well as to deliver chemotherapeutic agents for treating cancer.

Development and Characterization of Methylene Blue Oleate Salt-Loaded Polymeric Nanoparticles and their Potential Application as a Treatment for Glioblastoma.

Glioblastoma (GBM) is an aggressive, grade IV brain tumor that develops from astrocytes located within the cerebrum, resulting in poor prognosis and survival rates following an accepted treatment regimen of surgery, radiation, and temozolomide. Thus, development of new therapeutics is necessary. During the last two decades, methylene blue (MB) has received increased attention as a potential neurotherapeutic due to its duality in brain cancers and neurodegenerative diseases. While MB is capable of easily permeating the blood-brain barrier, its therapeutic concentrations in GBM are known to induce off-target cytotoxicity and thus, another mode of drug delivery must be considered. To this end, encapsulation of formerly unusable compounds into nanoparticles (NPs) made from the biodegradable/biocompatible, FDA approved co-polymer poly (lactide-co-glycolide) (PLGA) has been more commonplace when developing novel therapeutics. In this study, we formulated and characterized Pluronic F68-coated PLGA NPs containing a sodium oleate conjugate of MB (MBOS) via solvent displacement. Conjugation of sodium oleate to MB was shown to reduce its release from PLGA NPs compared to unmodified MB, leading to potential improvements in drug accumulation and therapeutic effectiveness. Our drug-loaded NP preparations, which were ~170 nm in size and had drug loading values of ~2%, were shown to reduce cell viability and cell compartment-specific, as well as overall cell, functions equivalenty, if not more so, when compared to free drug in two GBM cell lines. Following bio-distribution analysis of free MBOS compared to its nano-encapsulated counterpart, drug-loaded NPs were shown to more effectively permeate the BBB, which could lead to improvements in therapeutic effectiveness upon further examination in a tumor-bearing mouse model. Based on these results, we believe that the further development and eventual utilization of this nanoformulation could lead to an effective GBM therapy that could extend patient survival rates.

Annexin II expression is regulated during mammalian cell cycle.

Annexin II is a substrate for oncogene and growth factor-associated protein-tyrosine kinases. Elevated expression of annexin II is seen in different types of cancers and recent evidence indicates a role for annexin II in DNA synthesis and cell proliferation. In this report we show that the level of annexin II is subject to cell cycle regulation. Chinese hamster ovary cells were selected without the use of drugs, by the mitotic cell selection technique. As the mitotic cells progressed synchronously through the cell cycle, we determined the steady-state levels of annexin II mRNA and protein. The half-life of annexin II mRNA was approximately 2 h as measured by pulse-chase and ribonuclease protection analyses. Steady-state levels of both annexin II mRNA and protein were high in mitotic cells. As the cells divided and entered G1, there was a reduction in the levels of both annexin II mRNA and protein. New synthesis of annexin II mRNA and protein occurred in early G1 and maximal expression of annexin II mRNA and protein occurred as the cells entered S-phase. A gradual reduction in steady-state levels of annexin II mRNA and protein occurred as cells progressed through S-phase. Similar results were obtained in HeLa cells. In HeLa cells, collected at various cell cycle stages by countercurrent centrifugal elutriation, we observed peak expression of annexin II in G1-S and S-G2 cells. We conclude from our results that annexin II expression is regulated during the mammalian cell cycle.

Suramin affects DNA synthesis in HeLa cells by inhibition of DNA polymerases.

Suramin, a polysulfonated naphthylurea widely used in the treatment of trypanosomiasis and onchocerciasis, is currently being investigated as an antitumor agent for the treatment of advanced cancer. Suramin exerts a wide variety of biological effects. We have shown that suramin inhibits cell proliferation and DNA synthesis in cultured HeLa cells. The replication in vitro of SV40 DNA is completely abolished by 40 microM suramin. The inhibition of DNA replication is due to inhibition of DNA polymerases alpha and delta, the replicative enzymes in eukaryotic cells. DNA polymerase alpha is sensitive to lower concentrations of suramin [concentration to achieve 50% inhibition (IC50) of 8 microM] than is DNA polymerase delta (IC50 36 microM), whereas DNA polymerase beta is relatively insensitive to the drug (IC50 of 90 microM). Suramin inhibits other replicative DNA polymerases such as Escherichia coli polymerase I (Klenow fragment) and Thermus aquaticus polymerase. Suramin is noncompetitive with both substrate deoxyribonucleotides and template-primers with respect to DNA polymerase inhibition. Much lower concentrations (8-30 microM) of the drug are required for 50% inhibition of DNA polymerases than for 50% inhibition of other enzymes such as protein kinase C and reverse transcriptase. These results show an important biological effect of this drug and indicate the need for more studies before its clinical use as an antitumor agent.

Enhanced levels of annexins in pancreatic carcinoma cells of Syrian hamsters and their intrapancreatic allografts.

Annexins are a family of calcium- and phospholipid-binding proteins related by amino acid sequence homology. Annexins I and II are substrates for protein tyrosine kinases. Recent investigations have revealed a possible involvement of annexins I and II in mitogenic signal transduction and cell proliferation. To investigate further the involvement of annexins in cell proliferation, we measured the levels of annexins I and II and the enzyme 3-phosphoglycerate kinase (PGK) (annexin II and PGK are components of the primer recognition protein complex) in normal Syrian hamster pancreas, three hamster pancreatic ductal carcinoma cell lines, and allografts of the three cell lines into hamster pancreas. All three carcinoma cell lines had 5-8-fold higher levels of annexin II compared to normal pancreas. An inverse relationship was seen between level of annexin II and the doubling time of the cell culture. In intrapancreatic allografts, annexin II levels were 3-6-fold higher than in normal pancreas. Annexin I levels were 2-3-fold higher in the allografts. Significant increases (5-6-fold) in specific activity of PGK were seen in all allografts examined. However, the level of PGK, as measured by immunoblotting, was not significantly altered. Immunohistochemical staining revealed heterogeneity in the reactivity of the antiannexin and anti-PGK antibodies with tumor cells. Strikingly, the reactivity and staining intensity were greater in the proliferating regions of the primary tumors and in the metastatic foci. Mitotic cells were either unstained or very weakly stained. We conclude from these findings that annexin II and PGK, as primer recognition proteins, may have a role in cell proliferation.

Altered expression of annexin II in human B-cell lymphoma cell lines.

Annexin II is a growth-regulated gene, whose expression is significantly increased in various human cancers. We examined annexin II expression in II human B-cell lymphoma cell lines and in normal B-cells. Wide variation was observed in the levels of annexin II in these cell lines. Annexin II overexpression was observed in 5 cell lines, while significantly reduced expression was observed in Raji, OMA-BL-1 and REH cell lines. Analysis of the annexin II gene, mRNA and protein in Raji and OMA-BL-1 cell lines indicated that annexin II gene was unaltered and that a low level of annexin II transcripts are produced in these cells. Down-regulation of annexin II expression was at the transcriptional level, and no reexpression of annexin II was observed after treatment of cells with demethylating agents. Thus methylation of the annexin II gene does not appear to be responsible for annexin II down-regulation. A slow migrating altered form of annexin II was detected in Raji and OMA-BL-1 cells, which was detected with the anti-chicken annexin II antiserum, but not with the anti-human annexin II antiserum. The slow migrating annexin II species was found to be sensitive to dephosphorylation by calf intestinal alkaline phosphatase, resulting in reduction of the size of the protein on SDS-polyacrylamide gels. The phosphorylated annexin II was also observed in nuclear extracts of human K562 and HeLa cells. Thus, Raji and OMA-BL-1 cells exclusively produce a phosphorylated form of annexin II, and phosphorylated annexin II may be important for cell survival and proliferation.

Annexin I degradation in bronchoalveolar lavage fluids from healthy smokers: a possible mechanism of inflammation.

Annexin I is a glucocorticoid-inducible, phospholipase A2-inhibitory protein and is proposed to have an anti-inflammatory role. Although annexin I is a cytosolic protein, it is found extracellularly in secreted fluids such as semen. We have examined the expression of annexin I in bronchoalveolar lavage fluids (BALF) from smokers and nonsmokers to investigate the role of annexin I in the airway. We find that annexin I is secreted in BALF. This secretion is not due to cell death or damage, because a cytosolic protein, 3-phosphoglycerate kinase, is not seen in BALF. We observed that BALF from smokers (n = 10) had high protein concentrations as compared with BALF from nonsmokers (n = 11). Annexin I levels were higher in BALF from smokers compared with nonsmokers. However, in smokers, annexin I was exclusively found in the Mr 34,000 form that lacks the Mr 3,000 N-terminal anti-inflammatory peptide. In nonsmokers, both the Mr 37,000 native annexin I and the Mr 34,000 proteolytically cleaved form are present, with the Mr 37,000 form being most abundant. The NH2-terminal Mr 3,000 peptide of annexin I exhibits anti-inflammatory actions (G. Cirino et al, Br. J. Pharmacol., 108: 573-574, 1993). Previous studies have implicated neutrophil elastase as the protease cleaving annexin I to the Mr 34,000 protein. We observed increased elastase levels in BALF from smokers. However, we find no correlation between bronchial sample percent of neutrophils in BALF and the relative amount of the Mr 34,000 band generated. Our data clearly demonstrate that annexin I is degraded in BALF from smokers, and we propose that proteolytic cleavage of annexin I in BALF from smokers may be a mechanism by which polymorphonuclear neutrophils infiltrate sites of inflammation; thus, inactivation of annexin I in smokers' lungs may lead to chronic and uncontrolled inflammation.

Expression of 72 kDa and 92 kDa type IV collagenases from human giant-cell tumor of bone.

Basement membrane forms widespread barriers to tumor invasion. It has been shown that tumor-secreted, basement membrane-degrading enzymes, namely metalloproteinases (MMPs) play an important role in tumor invasion and metastasis. In this study, we determined the enzymatic activity, content, and mRNA of both the 72 kDa (MMP-2) and 92 kDa (MMP-9) MMPs in primary cultures of human giant-cell tumor of bone (GCT) in vitro and in tissue extracts (in vivo). Gelatin zymography showed the presence of lytic bands at M(r) 121,000, 92,000, and 72,000, and these enzymatic activities were inhibited by EDTA, an inhibitor of MMPs. Western blots with antibodies specific for MMP-2 and MMP-9 confirmed the presence of MMP-2 and MMP-9 both in vitro and in vivo, but GCT cells at late passage showed only MMP-2. Northern blots using labeled cDNA probes specific for these molecules revealed the presence of 3.1 kb transcript for MMP-2 and a 2.9 kb transcript for MMP-9. Using specific antibodies to 72 kDa and 92 kDa type IV collagenases, we studied their cellular distribution by immunohistochemical means. Stronger immunoreactivity was found for 92 kDa type IV collagenase than 72 kDa type IV collagenase in the giant cells. It appears, therefore, that MMP-9 may play an important role in the malignant behavior of GCTs and suggests a potential therapeutic role for protease inhibitors in attempting to minimize the invasive behavior of GCTs.

Tissue angiotensin I-converting enzyme activity in aging hamsters with and without cardiomyopathy.

The purpose of this study was to determine tissue angiotensin I-converting enzyme (ACE) activity in aging hamsters with and without cardiomyopathy, and the factors that regulate its activity in vitro. We found that ACE activity was significantly increased in the heart and significantly decreased in the lung of aging hamsters with hereditary cardiomyopathy in comparison to age/genetically-matched controls (P < 0.05). Kidney and cheek pouch ACE activity was similar in both groups. Lisinopril inhibition curves of tissue ACE activity were similar in aging hamsters with and without cardiomyopathy. In both groups, tissue ACE activity was dependent on chloride anion concentration in the assay buffer. Substituting citrate for chloride abrogated, in part, this response. We conclude that cardiomyopathy is associated with significant changes in cardiac and lung ACE activity in aging hamsters in comparison to age/genetically-matched controls. However, regulation of tissue ACE activity in vitro is similar in both groups.

Detection of secreted and intracellular annexin II by a radioimmunoassay.

A simple radioimmunoassay for detection of secreted and intracellular annexin II in human cells is presented. Annexin II is a multifunctional protein in human cells and may have a role in several types of cancers. No enzymatic activity has been associated with the protein, thus making its detection difficult. Using purified annexin II from human placenta, we have developed a sensitive radioimmunoassay protocol. A linear response was observed up to a concentration of 0.5 microgram purified protein in the assay. Using this radioimmunoassay protocol, annexin II can be detected in undiluted clinical human samples such as bronchoalveolar lavages and various tissue extracts. We demonstrate the applicability of this technique to measure intracellular annexin II in extracts of a human adenocarcinoma cell line (HeLa) and secreted annexin II from bronchoalveolar lavage fluid from a human patient. Using HeLa cell extracts and BAL, we observed a linear response with up to 10 micrograms total protein in the assay. We further demonstrate the applicability of this technique to measure differences in intracellular and secreted annexin II in the human pancreatic adenocarcinoma cell lines CD-11, CD-18 and Capan-2. While CD-11 and CD-18 do not secrete annexin II, the cell line Capan-2 secretes high levels of the protein.

Increased tissue neutral endopeptidase 24.11 activity in spontaneously hypertensive hamsters.

The purpose of this study was to determine whether tissue neutral endopeptidase (NEP) 24.11 activity, a membrane-bound metalloenzyme widely distributed in the peripheral circulation that cleaves and inactivates vasodilator peptides, is increased in spontaneously hypertensive hamsters relative to genetically/age-matched normotensive hamsters. Mean arterial pressure and heart rate were 163 +/- 11 mm Hg and 312 +/- 7 beats/min in spontaneously hypertensive hamsters and 99 +/- 3 mm Hg and 302 +/- 10 beats/min in normotensive hamsters, respectively (mean +/- SEM). NEP 24.11 activity is significantly increased in the kidney, cheek pouch, and spinotrapezius muscle, and significantly decreased in the heart and aorta of spontaneously hypertensive hamsters relative to controls (P < .05). Lung and brain NEP 24.11 activity is similar in both groups. Renal NEP 24.11 activity increases and to a similar extent in spontaneously hypertensive and normotensive hamsters as chloride anion concentration in the assay buffer is increased. Substituting citrate for chloride anion significantly attenuates renal NEP 24.11 activity. Taken together, these data indicate that NEP 24.11 activity in spontaneously hypertensive hamsters is increased in two organs that contribute appreciably to peripheral vascular resistance, skeletal muscle, and kidney. We suggest that the spontaneously hypertensive hamster is a suitable model to study the role of skeletal muscle and renal NEP 24.11 in regulating vasomotor tone in essential hypertension.

Loop diuretics attenuate bradykinin-induced increase in clearance of macromolecules in the oral mucosa.

The purpose of this study was to determine whether loop diuretics attenuate bradykinin-induced increase in clearance of macromolecules in the oral mucosa in situ and, if so, to start to determine the mechanisms that mediated these responses. By using intravital microscopy, we found that bradykinin induced a significant concentration-dependent increase in fluorescein isothiocyanate-labeled dextran (mol mass 70 kDa) leaky site formation in the hamster cheek pouch. These responses were significantly attenuated by topical application of two structurally distinct loop diuretics, furosemide and ethacrynic acid, onto the cheek pouch (P < 0.05). Hydrochlorothiazide, a nonloop diuretic, had no significant effects on bradykinin-induced responses. Furosemide had no significant effects on adenosine-induced leaky site formation. Application of bradykinin after furosemide, but not after hydrochlorothiazide, was associated with a significant concentration-dependent decrease in bradykinin-like immunoreactivity in the cheek pouch suffusate (P < 0.05). Prostaglandins and changes in vasomotor tone did not modulate the effects of furosemide on bradykinin-induced responses. These data indicate that loop diuretics attenuate bradykinin-induced increase in clearance of macromolecules in the oral mucosa in a specific fashion, probably by amplifying local bradykinin catabolism. We suggest that topical loop diuretics could be useful in the treatment of oral mucosa inflammation elicited by bradykinin.

Diadenosine polyphosphates: their biological and pharmacological significance.

Diadenosine polyphosphates are members of a group of dinucleoside polyphosphates that are ubiquitous in bacteria to mammals. In recent years, the diadenosine polyphosphates have received considerable attention in view of their multiple biological activities and potential pharmacological activities. Diadenosine polyphosphates have been identified as modulators of cardiovascular and neurotransmitter-like activities in recent years, besides their previously described role in cell proliferation and as signal molecules when cells are undergoing stress. Diadenosine polyphosphates and their synthetic analogues are being evaluated for their potential as pharmacological agents. This article discusses the various biological functions and physiological significance of the diadenosine polyphosphates.

Androgen signaling and post-transcriptional downregulation of Bcl-2 in androgen-unresponsive prostate cancer.

We previously characterized the LNCaP prostate cancer progression model and showed that despite loss of Bcl-2 protein in the androgen-unresponsive LNCaP-unresponsive (UR) cells, these cells maintained an increased resistance to the induction of apoptosis. Since the loss of Bcl-2 protein coincided with the progression to androgen-unresponsiveness, we sought to determine if Bcl-2 expression was regulated through androgen signaling pathways. LNCaP-responsive (R) and -UR cells grown in charcoal-stripped serum conditions for 3 months differentiated to a neuroendocrine (NE)-like morphology. Under these conditions, LNCaP-UR cells regained Bcl-2 protein expression, and LNCaP-R cells overexpressed Bcl-2. Chronic exposure to casodex resulted in differentiation of both LNCaP-R and -UR cells to the NE-type morphology accompanied by a marked downregulation of Bcl-2 protein, while Bax protein levels were unchanged. Downregulation of Bcl-2 was post-transcriptional since Bcl-2 message levels were unchanged in LNCaP cells treated with casodex. These data suggest that Bcl-2 is post-transcriptionally modulated by androgen signaling pathways in LNCaP cells.

Regulation of Bcl-2 during androgen-unresponsive progression of prostate cancer.

The progression of prostate cancer from androgen-responsive to an androgen-unresponsive state remains the greatest obstacle in the treatment of this disease. Androgen-unresponsive prostate cancer is highly resistant to chemotherapy and radiation treatment that kill cells by the induction of apoptosis. Elucidating the molecular mechanisms of apoptosis regulation in prostate cancer can be useful in the development of new strategies for effective therapy of androgen-unresponsive cancer. We analyzed the Bcl-2 family of apoptosis regulators using various passages of the LNCaP prostate cancer cell line, which serve as an in vitro model for the progression of prostate cancer from androgen-responsive to androgen-unresponsive. In our model, progressively higher passages of LNCaP cells represent the progression to androgen-unresponsiveness. We examined the basal mRNA expression of the Bcl-2 family of apoptosis regulators. Under normal growth conditions, both androgen-responsive and androgen-unresponsive LNCaP cells express the Bcl-2 family of genes at similar levels. Western blot analysis showed the presence of Bcl-2 protein in androgen-responsive cells but not in androgen-unresponsive cells. Both androgen-responsive and androgen-unresponsive cells expressed Bax protein at similar levels. When exposed to oxidative stress, androgen-responsive cells underwent apoptosis but androgen-unresponsive cells exhibited resistance suggesting that the progression to androgen-unresponsiveness was associated with altered regulation of apoptosis. Treatment with paclitaxel or sodium butyrate induced apoptosis in both androgen-responsive and androgen-unresponsive cells suggesting that the apoptotic machinery is still intact in androgen-unresponsive LNCaP cells.

Combinatorial nanoparticles for cancer diagnosis and therapy.

Nanotechnology when engineered together with biotechnology opens a fascinating field with applications in diverse areas such as drug targeting and delivery, medical imaging, biosensing, biomaterials and nanotechnology. Conjugating nanoparticles with biomolecules like QD-herceptin conjugates or QD-aptamer (Apt)-DOX conjugates provides many opportunities for improving many of the current challenges in cancer diagnosis and therapy. This paper reviews combinatorial nanoparticles designed and formulated for cancer imaging and therapy, including inorganic nanoparticles (quantum dots, iron oxide particles, gold nanoparticles and silica and carbon nanoparticles), polymeric nanoparticles (PLGA, PLGA-PEG, PAMAM), liposomes and lipid nanoparticles. These nanoparticles are multifunctional in nature and combine two or more functions like targeting, imaging and therapy. In this review, we have classified these combinatorial targeted nanoparticles into inorganic, polymeric and liposome based nanosystems.

Novel gene C17orf37 in 17q12 amplicon promotes migration and invasion of prostate cancer cells.

C17orf37/MGC14832, a novel gene located on human chromosome 17q12 in the ERBB2 amplicon, is abundantly expressed in breast cancer. C17orf37 expression has been reported to positively correlate with grade and stage of cancer progression; however the functional significance of C17orf37 overexpression in cancer biology is not known. Here, we show that C17orf37 is highly expressed in prostate cancer cell lines and tumors, compared to minimal expression in normal prostate cells and tissues. Cellular localization studies by confocal and total internal reflection fluorescence microscopy revealed predominant expression of C17orf37 in the cytosol with intense staining in the membrane of prostate cancer cells. RNA-interference-mediated downregulation of C17orf37 resulted in decreased migration and invasion of DU-145 prostate cancer cells, and suppressed the DNA-binding activity of nuclear factor-kappaB (NF-kappaB) transcription factor resulting in reduced expression of downstream target genes matrix metalloproteinase 9, urokinase plasminogen activator and vascular endothelial growth factor. Phosphorylation of PKB/Akt was also reduced upon C17orf37 downregulation, suggesting C17orf37 acts as a signaling molecule that increases invasive potential of prostate cancer cells by NF-kappaB-mediated downstream target genes. Our data strongly suggest C17orf37 overexpression in prostate cancer functionally enhances migration and invasion of tumor cells, and is an important target for cancer therapy.

Phosphorylation of HeLa cell multiprotein DNA polymerase alpha complex: impact on activity and partial purification of the associated kinase.

Phosphorylation is a major post-translational regulatory mechanism and plays a key role in transduction of mitogenic signals in cell proliferation. The role of phosphorylation and dephosphorylation in regulating the activities of a multiprotein DNA polymerase alpha complex was examined. Treatment of the HeLa cell multiprotein DNA polymerase alpha with calf intestinal alkaline phosphatase resulted in the inactivation of DNA polymerase alpha and DNA primase but had no effect on deoxyribonuclease- and primer-recognition proteins. A protein kinase co-purified with the multiprotein DNA polymerase alpha and was partially purified from HeLa cells. The partially purified kinase was active in phosphorylating dephosphorylated polymerase alpha and used casein and histones as exogenous substrates. This study demonstrates that phosphorylation-dephosphorylation may have modulated the activities of DNA replicative enzymes and suggests a role for specific phosphatases and kinases in this process.

Involvement of annexin II in DNA replication: evidence from cell-free extracts of Xenopus eggs.

Cell-free extracts of Xenopus eggs efficiently initiate and complete semiconservative DNA replication of exogenously added plasmid DNA. DNA replication in such extracts can be neutralized by a monoclonal antibody (D1/274.5) against human annexin II. Specific immunodepletion of Xenopus annexin II from the egg extracts results in loss of DNA replicative ability. Immunodepletion of annexin II does not prevent nuclear assembly, a stringent requirement for DNA synthesis on exogenous DNA in this system. Replicative ability can be restored to the immunodepleted extracts by the addition of purified human annexin II. These results demonstrate that annexin II is involved in chromosomal DNA replication and has a role in the cell cycle of higher eukaryotes.