Investigation of a Novel Decision Support Metric for Head and Neck Adaptive Radiation Therapy Using a Real-Time In Vivo Portal Dosimetry System.

In adaptive radiation therapy of head and neck cancer, any significant anatomical changes observed are used to adapt the treatment plan to maintain target coverage without elevating the risk of xerostomia. However, the additional resources required for adaptive radiation therapy pose a challenge for broad-based implementation. It is hypothesized that a change in transit fluence is associated with volumetric change in the vicinity of the target and therefore can be used as a decision support metric for adaptive radiation therapy. This was evaluated by comparing the fluence with volumetric changes in 12 patients. Transit fluence was measured by an in vivo portal dosimetry system. Weekly cone beam computed tomography was used to determine volume change in the rectangular region of interest from condyloid process to C6. The integrated transit fluence through the region of interest on the day of the cone beam computed tomography scan was calculated with the first treatment as the baseline. The correlation between fluence change and volume change was determined. A logistic regression model was also used to associate the 5% region of interest volume reduction replanning trigger point and the fluence change. The model was assessed by a chi-square test. The area under the receiver-operating characteristic curve was also determined. A total of 46 pairs of measurements were obtained. The correlation between fluence and volumetric changes was found to be -0.776 (P value <.001). The negative correlation is attributed to the increase in the photon fluence transport resulting from the volume reduction. The chi-square of the logistic regression was found to be 17.4 (P value <.001). The area under the receiver-operating characteristic curve was found to be 0.88. Results indicate the change in transit fluence, which can be measured without consuming clinical resources or requiring additional time in the treatment room, can be used as a decision support metric for adaptive therapy.

Cell cycle effects of IL-10 on malignant B-1 cells.

IL-10 is overexpressed in human chronic lymphocytic leukemia (CLL), and is an autocrine growth factor involved in the development of malignant B1 clones in NZB mice, a murine model for CLL. Antisense IL-10 oligonucleotide treatment induces apoptosis and cell cycle disruption in these cells both in vitro and in vivo. In addition, NZB IL-10 knock-out mice fail to develop the B-1 clones. Dampening of IL-10 protein production via antisense IL-10 oligonucleotide treatment is correlated with decreased p27/Kip1 protein expression which results in increased cyclin D2, cyclin E and cyclin A associated kinase activity. The action of the antisense oligonucleotides is through alterations in cell cycle regulation, resulting in accelerated cell cycle progression, a G2/M block which culminates in apoptosis induction in the malignant cells. This implies that the role of IL-10 as an autocrine growth factor in malignant B-1 cells lies in its ability to inhibit apoptosis induction through the maintenance of sustainable cell cycle progression in malignant cells.

The growth-regulatory role of B-cell-specific activator protein in NZB malignant B-1 cells.

The transcription factor B-cell-specific activator protein (BSAP) plays an important role in B-cell development. We explored the involvement of BSAP in the growth regulation of malignant B-1 cells derived from the NZB murine model of human chronic lymphocytic leukemia. BSAP protein was found in normal B-2 cells, elevated in normal B-1 cells, and highest in NZB malignant B-1 cells. When these malignant B-1 cells were treated with antisense oligonucleotides for BSAP, their growth was inhibited with a G2/M phase arrest. In contrast, B cell lines that did not appear to be of B-1 origin (IgG+/B220+/BSAPlow) were unaffected by treatment with antisense BSAP oligonucleotides. Centrifugal elutriation experiments showed that BSAP mRNA was expressed at the highest levels in the G2/M phases in malignant B-1 cells. Treatment with demecolcine (Colcemid), a known mitotic blocker, resulted in a decrease in the level of BSAP gene expression in malignant B-1 cells, further demonstrating links between BSAP expression and successful G2/M transition in the cell cycle. These data suggest a correlation between BSAP and the development of B-1 malignancy, perhaps through the regulation of cell-cycle progression.

Overexpression of p21(waf1) in human T-cell lymphotropic virus type 1-infected cells and its association with cyclin A/cdk2.

Human T-cell lymphotropic virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). T-cell transformation is mainly due to the actions of the viral phosphoprotein Tax. Tax interacts with multiple transcriptional factors, aiding the transcription of many cellular genes. Here, we report that the cyclin-dependent kinase inhibitor p21/waf1 is overexpressed in all HTLV-1-infected cell lines tested as well as in ATL and HAM/TSP patient samples. Tax was found to be able to transactivate the endogenous p21/waf1 promoter, as detected by RNase protection, as well as activate a series of wild-type and 5'-deletion constructs linked to a luciferase reporter cassette. Wild-type but not a mutant form of Tax (M47) transactivated the p21/waf1 promoter in a p53-independent manner and utilized a minimal promoter that contained E2A and TATA box sequences. The p21/waf1 protein was reproducibly observed to be complexed with cyclin A/cdk2 and not with any other known G(1), S, or G(2)/M cyclins. Functionally, the association of p21/cyclin A/cdk2 decreased histone H1 phosphorylation in vitro, as observed in immunoprecipitations followed by kinase assays, and affected other substrates, such as the C terminus of Rb protein involved in c-Abl and histone deacetylase-1 (HDAC1) regulation. Interestingly, upon the use of a stress signal, such as gamma-irradiation, we found that the p21/cyclin A/cdk2 complex was able to block all known phosphorylation sites on the Rb molecule. Finally, using elutriated cell cycle fractions and a stress signal, we observed that the HTLV-1-infected T cells containing wild-type Tax, which had been in early or mid-G(1) phase prior to gamma-irradiation, arrested in G(1) and did not undergo apoptosis. This may be an important mechanism for an oncogenic virus such as HTLV-1 to stop the host at the G(1)/S boundary and to repair the damaged DNA upon injury, prior to S-phase entry.

p53/56(lyn) antisense shifts the 1,25-dihydroxyvitamin D3-induced G1/S block in HL60 cells to S phase.

p53/56(lyn) is a member of the src family that is predominantly expressed in hematopoietic cells and is thought to play a role in cellular proliferation. In this study, we demonstrate the participation of p53/56(lyn) in 1,25-dihydroxyvitamin D(3) (1, 25D(3))-induced growth arrest in HL60 cells. We show that the mRNA and protein levels of p53/56(lyn) are markedly elevated after 1, 25D(3) treatment, which is accompanied by an increase of p53/56(lyn) kinase activity. We also demonstrate that treatment with p53/56(lyn) antisense oligodeoxynucleotides reverses the 1,25D(3)-induced G1/S block, and results in an accumulation of cells with S-phase DNA content. BrdU pulse-chase experiments reveal that this accumulation results from an increased proportion of cells actively synthesizing DNA, which are inhibited from exiting the S-phase compartment. These results indicate that upregulation of p53/56(lyn) contributes significantly to the G1/S growth arrest induced by 1,25D(3) in HL60 cells and thus its activation may be a desirable outcome of chemotherapeutic regimens.

Differentiation-related mechanisms which suppress DNA replication.

Differentiation of mammalian cells implies cessation of DNA replication and cell proliferation; the potential controls of this coupling are examined here. It is clear that the known or proposed mechanisms of down-regulation of replicative cellular activities vary in different lineages of cell differentiation, and occur in all phases of the cell cycle. In G1 these regulators include p21/Cip1 or p27/Kip1, pRb, and p53; the novel, recently reported mechanisms of their action are summarized. In S phase the availability of nucleotide precursors, the origin recognition complex (ORC), and other replication proteins may be important in differentiation, and in G2 phase the cdc2/cyclin B complex and replication licensing factors determine normal G2 traverse versus an arrest or polyploidisation. Other replication-related mechanisms include transcription factors, e.g., Sp1, telomerase, and nuclear matrix changes. Thus, differentiation alters the activity not only of the various checkpoint proteins, but also of the components of the replicative machinery itself.

Protein phosphorylation associated with epipodophyllotoxin-induced apoptosis of lymphoid cells: role of a serine/threonine protein kinase.

We have previously shown that apoptosis induced in thymocytes by dexamethasone or teniposide (VM-26) could be inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and sangivamycin, both relatively specific inhibitors for protein kinase C, but not by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a more specific inhibitor for cAMP-dependent protein kinases. Apoptosis in this model system was not blocked by EGTA and no increase in cytosolic Ca2+ was observed during apoptosis induced by either dexamethasone or VM-26, suggesting that this kinase was Ca2+-independent. In the present study, we demonstrate that addition of 10 microM sangivamycin to thymocyte cultures up to 2 h after addition of either inducer resulted in virtually complete inhibition of apoptosis. Addition of 10 microM sangivamycin at 3 or 4 h after addition of inducer resulted in partial inhibition of apoptosis. Computerized image analysis of two-dimensional PAGE analyses of whole-cell lysates demonstrated that treatment of mouse thymocytes with VM-26 resulted in a limited number of de novo phosphorylation events within 1 h of treatment. The most prominent phosphorylation events associated with VM-26-induced apoptosis were that two intracellular protein species (Protein 1: m.w. = 22.9 kDa, pI, 5.11; and Protein 2: m.w. = 22.9 kDa, pI, 4.98). Similar phosphorylation events were seen in cells treated with dexamethasone. Finally, Western blot analysis suggests that de novo protein phosphorylation induced by VM-26 is on serine/threonine residues. These results provide further evidence that the mechanism of VM-26-induced apoptosis of murine thymocytes involves the action of one or more serine/threonine kinases.

Retinoblastoma protein-overexpressing HL60 cells resistant to 1,25-dihydroxyvitamin D3 display increased CDK2 and CDK6 activity and shortened G1 phase.

Drug resistance that occurs during cancer chemotherapy has been a major problem in controlling neoplastic progression. To study the cellular mechanisms of acquired drug resistance we developed 1,25-dihydroxyvitamin D3 (1,25D3)-resistant sublines of promyelocytic leukemia HL60 cells which have increased proliferation rates (Exp. Cell Res., 224, 312, 1996; Cancer Res., 50, 5513, 1996). We report here that the resistant sublines display varying degrees of shortening of the G1 phase as compared to the parental HL60-G cells. Protein levels of cyclins E, D1, D2 and D3 are elevated in these resistant cell lines, and cyclin D1 is especially high in 40AF cells, which has the shortest G1 length. The protein levels of cyclin-dependent kinase (Cdk)2, Cdk4 and Cdk6 are not altered in the resistant sublines. Both Cdk2 and Cdk6-associated kinase activites are increased in the resistant sublines, but not Cdk4 kinase activity. Protein levels of p27Kip1 are not consistently altered in the resistant sublines as compared to the parental HL60-G cells, but are reduced relative to HL60-G cells arrested by 96 h treatment with 1,25D3. Interestingly, the resistant cell lines constitutively express high levels of retinoblastoma protein (pRb), and pRb is highly phosphorylated, indicating that the G1 cyclin/Cdk complexes in the resistant cells are physiologically active. The results suggest that the increased activity of cyclin D/Cdk6, and perhaps cyclin E/Cdk2, lead to rapid hyperphosphorylation of pRb and consequently a shorter early G1 phase, and that in the resistant cells the increased ratio of cyclin E to p27Kip1 results in activation of Cdk2 and contributes to the abrogation of the 1,25D3-induced block to the S phase entry. Additionally, it is apparent that constitutively increased levels of pRb are compatible with increased rates of cell proliferation.

Inhibition of T cell activation by an autoantibody induced by murine retrovirus infection.

This report describes a murine monoclonal IgM antibody, 6E3.C4, induced by retrovirus infection of BALB/ c-H-2b mice which inhibits mitogen stimulation of both mouse and human lymphocytes in vitro. The molecule bound by this antibody appears to be an activation antigen since binding is upregulated by mitogen stimulation. Analysis of the epitope bound by mAb 6E3.C4 revealed that it is associated with a 52-kDa protein with a pI of approximately 5.7 as determined by Western blot analysis. A protein expressing this or a cross-reactive epitope was isolated and determined to be alpha-tubulin by amino acid sequencing. Reactivity with purified alpha-tubulin confirms this identification. These findings suggest a potential role for a cell surface molecule that is either alpha-tubulin or a cross-reactive molecule in the activation processes of T cells.

Regulation of DNA replication initiation in mammalian lymphocyte systems.

The control of DNA replication is central to the control of cell proliferation, and defects in S phase regulation have been implicated in senescence and neoplasia. To examine the regulation of DNA replication in lymphocytes, an in vitro system was developed in which lymphocyte derived proteins could regulate the initiation of DNA replication in isolated quiescent nuclei. Cytosolic extracts from mitogen or IL-2 activated lymphocytes as well as lymphoblastoid cell lines produce a factor (Activator of DNA replication; ADR) that can induce DNA synthesis in isolated quiescent nuclei, and DNA synthesis in this system is consistent with DNA replication and not repair. ADR activity is tightly associated with a protease activity and is not detectable in resting cells, but can be induced by a mechanism dependent on serine/threonine and tyrosine phosphorylation. Quiescent cells contain an ADR inhibitor which blocks DNA synthesis in isolated normal nuclei but not in nuclei from transformed cells, a potential factor in the uncontrolled proliferation of neoplastic cells. The control of cellular DNA replication is dependent on the interaction of origin sequences with specific replicative and regulatory proteins. However, mammalian origins of DNA replication are not well defined. Plasmids containing a replication origin within the human rRNA gene can act as replicative templates in our cell-free replication system, thus allowing a detailed molecular dissection of replication initiation in a completely human experimental system.

Modulation of topoisomerase activities by tumor necrosis factor.

A number of chemotherapeutic agents which inhibit the DNA topoisomerases markedly potentiate cell death mediated by tumor necrosis factor, suggesting a role for these enzymes in the TNF cytotoxic mechanism. To investigate this possibility, topoisomerase I and II activities were assayed following TNF addition to murine L929 cells. Topoisomerase I and II activities increased within 15 min of TNF addition and returned to baseline levels within 1 and 2 hr, respectively. The increases in both topoisomerase activities were blocked by H-7 (but not H-8) and similar increases were seen following PMA addition. However, concentrations of H-7 which blocked the increased topoisomerase activities had no effect on TNF cytotoxicity nor on the enhancement of TNF cytotoxicity by topoisomerase inhibitors. Thus, in these cells topoisomerase activities are directly modified by TNF during the initial phases of a cytotoxic response. However, neither TNF cytotoxicity nor the enhancement of TNF cytotoxicity by topoisomerase inhibitors appears to require the TNF-mediated increases in topoisomerase activities.

Characterization and purification of a macrophage-triggering factor produced in Mycoplasma arginini-infected L5178Y cell cultures.

The supernatant of Mycoplasma arginini-infected murine L5178Y T lymphoma cell cultures (SN-L51) synergizes with small concentrations of IFN-gamma to activate murine peritoneal, thioglycollate-elicited macrophages (M phi) to exhibit cytostatic activity against tumor cells. Treatment of M phi with IFN-gamma and SN-L51 sequentially, but not in the reverse order, activates M phi, which indicates that SN-L51 contains a M phi-triggering factor (MTF). MTF activity could be inhibited by small concentrations of prostaglandin E2, but not by polymyxin B. M phi activated by IFN-gamma plus MTF produce cytostatic effects on tumor cells through a nitric oxide-dependent pathway. MTF activity in SN-L51 is associated with infection of L5178Y cells by M. arginini. Mycoplasma-free L5178Y cells do not produce MTF activity, infection of these L5178Y cells with M. arginini generates the activity, and supernatants of pure M. arginini cultures contain MTF activity. MTF activity is thermostable and resistant to acid, dilute alkali, proteases, and nucleases. MTF was partially purified by ammonium sulfate precipitation, chromatography, electrophoresis, and electroelution. On 12.5% SDS-urea gels, MTF activity migrated with a molecular mass of 2.5 to 4 kDa. MTF activity and the silver staining of this band was resistant to proteinase K; however, Coomassie staining of this band was abolished by proteinase K. The combined data suggest that MTF is either a stable peptide or a peptide linked to lipid or carbohydrate.

Methylxanthines and calcium-mobilizing agents inhibit the expression of cytokine-inducible nitric oxide synthase and vascular cell adhesion molecule-1 in murine microvascular endothelial cells.

In response to exposure to the inflammatory cytokines tumor necrosis factor-alpha (TNF) and interferon-gamma (IFN-gamma), murine brain microvascular endothelial cells (MME) synthesize the cell surface molecule, vascular cell adhesion molecule-1 (VCAM-1), and the intracellular enzyme, inducible nitric oxide synthase (iNOS). However, iNOS synthesis requires the presence of both TNF and IFN-gamma, while VCAM-1 can be induced by either cytokine alone. We examined the induction of VCAM-1 and iNOS under a variety of conditions to better define the regulation of TNF and IFN-gamma signal transduction pathways in MME. We utilized the analysis of steady-state levels of iNOS mRNA as well as the measurement of MME-released NO-EDRF (nitric oxide as an endothelium-derived relaxing factor) activity and accumulation of nitrite in the culture medium to define iNOS expression and activity. VCAM-1 expression was determined by flow cytometric analysis. Our data indicate that low density lipoproteins inhibited cytokine-induced iNOS activity by affecting the steady-state levels of iNOS mRNA. Methylxanthines (caffeine and theophylline) as well as several calcium-mobilizing agents inhibited the expression/activity of both iNOS and VCAM-1 in MME. The effectiveness of these agents was dependent upon the degree of disruption in cell calcium homeostasis during cytokine treatment. Cells which had been pretreated with calcium-modulating drugs and then washed and allowed to return to normal calcium homeostasis showed little to no effect from these agents. In addition, our results suggest that NO produced by iNOS acts as a metabolic switch during inflammation by inhibiting oxidative phosphorylation and forcing vascular endothelial cells to temporarily utilize anaerobic energy metabolism.

In vitro replication of plasmids containing human ribosomal gene sequences: origin localization and dependence on an aprotinin-binding cytosolic protein.

We previously investigated the role of an aprotinin-binding protein (ADR) in the initiation of DNA replication in isolated quiescent nuclei. In the present study, we have used a cell-free DNA replication system to test the ability of plasmid vectors which contain sequences from the human ribosomal RNA gene to serve as replicative templates in vitro when exposed to ADR-containing preparations. Significant dTTP incorporation was seen using DNA from either a 7-kb sequence in the 5' spacer region (CHE) or a 7-kb sequence which begins near the end of the 28S coding region and extends into the 3' spacer region (ADBB), while sequences from other regions of the rRNA gene mediated little or no dTTP incorporation. The characteristics of plasmid-directed dTTP incorporation indicate that most incorporation is due to DNA replication and not repair or damage-initiated processes. To conclusively demonstrate origin-dependent replication in the plasmid system and to further map replication origins, an approach was developed using ddGTP to restrict the length of daughter strands followed by hybridization of these replication products to restriction fragments spanning the putative origin region. This approach allowed us to identify replication origin activity apart from parent strand repair or synthesis initiated at random damaged sites. One of the origins was localized to a 1375-bp fragment within the 5' spacer region, and this fragment contains sequences homologous to those found in other replication origins.

The mechanism of epipodophyllotoxin-induced thymocyte apoptosis: possible role of a novel Ca(2+)-independent protein kinase.

The epipodophyllotoxins, etoposide (VP-16) and teniposide (VM-26), inhibit topoisomerase II activity by stabilization of the cleavable complex between the enzyme and DNA and formation of protein-bound double-stranded DNA breaks. While it is thought that these agents are cytotoxic by preventing cells from completing the S phase or undergoing mitosis, recent evidence suggests that these agents are also potent inducers of programmed cell death or apoptosis in both normal and malignant cells. We have examined the intracellular pathway leading to epipodophyllotoxin-induced apoptosis in normal mouse thymocytes. Epipodophyllotoxin-induced apoptosis may proceed via a mechanism that is independent of inhibition of topoisomerase activity per se because novobiocin and coumermycin, which inhibit the ATPase subunit of topoisomerase II, were relatively inefficient inducers of apoptosis in these cells, under conditions where strong apoptosis by the epipodophyllotoxins and dexamethasone could be observed. In addition, camptothecin, which inhibits topoisomerase I by stabilization of the cleavable complex between that enzyme and DNA, was also a poor inducer of apoptosis in these cells. Our data suggest that epipodophyllotoxin-induced mouse thymocyte apoptosis, like that induced by dexamethasone, proceeds via a mechanism that involves protein kinase C (PKC) or a similar enzyme. Apoptosis induced by VM-26 or by dexamethasone was inhibited by 1-(5-isoquinolinylsulfonyl)-2- methylpiperazine dihydrochloride (H7), an inhibitor of both PKC and cAMP-dependent protein kinases, but was relatively unaffected by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a more specific inhibitor of cAMP-dependent protein kinases. A more specific inhibitor of PKC, sangivamycin, also inhibited both VM-26-induced and dexamethasone-induced apoptosis. Both VM-26- and dexamethasone-induced apoptosis were unaffected by EGTA, a calcium (Ca2+) chelator, under conditions that inhibited apoptosis induced by the Ca2+ ionophore A23187. Moreover, while strong increases in intracellular Ca2+ were observed in thymocytes treated with A23187, we failed to detect increases in intracellular Ca2+ in cells induced to apoptose with either VM-26 or dexamethasone within the first 2 hr of culture. These results suggest that in mouse thymocytes there are at least two intracellular pathways leading to apoptosis: one, utilized by glucocorticoid and the epipodophyllotoxins, that proceeds in the absence of detectable increases in intracellular Ca2+ and possibly requires a novel Ca(2+)-independent PKC-like enzyme and another, utilized by Ca2+ ionophores, that is at least partially dependent on increased intracellular Ca2+.

A cytosolic activator of DNA replication is tyrosine phosphorylated in its active form.

Cytosolic extracts from actively dividing lymphoid cells have been shown to induce DNA synthesis in isolated, quiescent nuclei. An initiating factor in such extracts (activator of DNA replication; ADR) is a > 90-kDa aprotinin-binding protein whose activity is inhibitable not only by aprotinin, but also by several other protease inhibitors as well. Although cytosol from non-proliferating lymphocytes is devoid of ADR activity, we have shown that these preparations can be induced to express ADR activity by brief exposure to a membrane-enriched fraction of spontaneously proliferating MOLT-4 cells via a kinase-dependent mechanism. In the present study, we examine the role of tyrosine kinases in this process. Three inhibitors of tyrosine kinases (genistein, kaempferol, and quercetin) can inhibit the in vitro generation of ADR activity. In vitro generation of ADR activity is associated with the de novo phosphorylation of several proteins, many of which are detectable using anti-phosphotyrosine monoclonal antibodies. ADR itself may be tyrosine phosphorylated in active form as immunoprecipitation using such monoclonal antibodies leads to the depletion of its activity. Moreover, immunoprecipitation results in the removal of several de novo tyrosine-phosphorylated proteins, including species at approximately 122, 105, 93, 86, 79, and 65 kDa. A subset of de novo-phosphorylated proteins, migrating at approximately 105, 93, and 70 kDa, also bound to aprotinin, suggesting that at least one of these proteins may represent ADR itself.

Characteristics of DNA replication in isolated nuclei initiated by an aprotinin-binding protein.

Isolated cell nuclei were used as the source of template DNA to investigate the role of a cytosolic aprotinin-binding protein (ADR) in the initiation of eukaryotic DNA replication. Computerized image cytometry demonstrated that the DNA content of individual nuclei increased significantly following incubation with ADR-containing preparations, and the extent of DNA synthesis is consistent with that allowed by the limiting concentration of dTTP. Thus, dTTP incorporation into isolated nuclei represents DNA synthesis and not parent strand repair. We found that dTTP incorporation into the isolated nuclei is dependent on DNA polymerase alpha (a principal polymerase in DNA replication) but that DNA polymerase beta (a principal polymerase in DNA repair processes) does not play a significant role in this system. Finally, neither aprotinin nor a previously described cytosolic ADR inhibitor can block the replication of nuclease-treated calf thymus DNA, while both strongly inhibit replication of DNA in isolated nuclei. This result, coupled with the relative ineffectiveness of nuclease-treated DNA compared with nuclear DNA to serve as a replicative template in this assay, argues against a significant contribution from repair or synthesis which initiates at a site of DNA damage. These data indicate that ADR-mediated incorporation of 3H-dTTP into isolated nuclei results from DNA replicative processes that are directly relevant to in vivo S phase events.

Inhibition of basal and tumor necrosis factor-enhanced binding of murine tumor cells to murine endothelium by transforming growth factor-beta 1.

The adherence of cells to microvascular endothelium is important in a number of processes, including inflammatory responses and metastasis. It has been demonstrated that in human models, cytokines such as TNF, IL-1, IFN-gamma increase the adhesiveness of endothelium for cells of the immune and inflammatory system by stimulating the expression of cell adhesion molecules on endothelial cell surfaces. We and others have shown similar cytokine-induced endothelial adhesiveness for tumor cells in murine and human models. In contrast to the effect of those modulators, transforming growth factor-beta (TGF-beta) has been shown to inhibit the binding of human neutrophils and T lymphocytes to human endothelium, although the mechanism of TGF-beta action remains unknown. Little is known about the effect of TGF-beta on tumor cell-endothelial interaction. In the present study, we demonstrate that TGF-beta inhibits basal and TNF-enhanced binding of murine P815 mastocytoma cells to murine microvascular endothelium (MME). The alterations in MME mediated by TGF-beta, also lead to the inhibition of adherence of murine splenocytes, thymocytes, and human lymphoblastoid cells but do not inhibit adherence of murine B16 melanoma cells. The effect of TGF-beta is transient and inhibition of the endothelial adhesive phenotype is strongest 12 to 24 h after addition of the factor to MME. The TGF-beta-mediated inhibition of P815 basal binding to endothelium is dependent on protein synthesis because cycloheximide reverses the TGF-beta effect. TGF-beta does not appear to activate classical signal transduction pathways. Inhibitors of G proteins do not abolish TGF-beta action, protein kinase C and protein kinase A activators elicit an effect opposite to that of the factor, TGF-beta does not increase intracellular cAMP levels, and finally calcium-mobilizing agents do not mimic, but rather inhibit the effect of TGF-beta. However, TGF-beta-mediated inhibition of both basal binding and TNF-enhanced P815 binding to MME is completely abolished in the presence of the protein phosphatase inhibitor okadaic acid which suggests that TGF-beta may elicit its effect by stimulating protein phosphatase activity.

Control of DNA replication in a transformed lymphoid cell line: coexistence of activator and inhibitor activities.

Proliferating lymphocytes contain an intracellular factor, ADR (activator of DNA replication), which can initiate DNA synthesis in isolated quiescent nuclei. Resting lymphocytes lack ADR activity and contain an intracellular inhibitory factor that suppresses DNA synthesis in normal but not transformed nuclei. In this study we describe a MOLT-4 subline that produces both the activator and inhibitory activities which can be separated by ammonium sulfate fractionation. The inhibitor is heat stable and inhibits ADR-mediated DNA replication in a dose-dependent manner. It does not inhibit DNA polymerase alpha activity. The inhibitor must be present at the initiation of DNA replication to be effective, as it loses most of its effectiveness if it is added after replication has begun. The presence of inhibitory activity in proliferating MOLT-4 cells, taken with the previous observation that inhibitor derived from normal resting cells does not affect DNA synthesis by MOLT-4 nuclei, suggests that failure of a down-regulating signal may play an important role in proliferative disorder.

Initiation of lymphocyte DNA synthesis.

The initiation of DNA replication in T lymphocytes appears to be regulated by two distinct activities: one associated with proliferation which mediates initiation, and another associated with quiescence which blocks initiation. Activated lymphocytes and proliferating lymphoid cell lines produce an activity, termed ADR, which can initiate DNA replication in isolated, quiescent nuclei. ADR is heat-labile, has protease activity or interacts closely with a protease, and is distinct from the DNA polymerases. ADR activity is absent in quiescent lymphocytes and appears in mitogen-stimulated lymphocytes after IL-2 binding. The generation of active ADR appears to be mediated by phosphorylation of a precursor which is present in resting cells. Nuclei from mitogen-unresponsive lymphocytes fail to initiate DNA replication in response to ADR, of potential importance in the age-related decline of immunity. Quiescent lymphocytes lack ADR and synthesize an ADR-inhibitory activity. The ADR inhibitor is a heat-stable protein which suppresses the initiation of DNA synthesis, but is ineffective at suppressing elongation once DNA strand replication has begun. Nuclei from several neoplastic cell lines fail to respond to the ADR inhibitor, which may play a role in the continuous proliferation of these cells. At least one of these neoplastic cell lines produces both ADR and an inhibitory factor. These findings suggest that the regulation of proliferation is dependent on the balance between activating and inhibitory pathways.