Alpha- and beta-adrenergic receptor (AR) protein expression is associated with poor clinical outcome in breast cancer: an immunohistochemical study.

Breast cancer mortality is frequently associated with metastatic disease. Metastasis models have shown adrenoceptor (AR) stimulation induces cell migration which is inhibited by adrenoceptor antagonist drugs. We investigated adrenoceptor protein expression in clinical breast tumours and its association with disease progression and prognosis. Immunohistochemistry on tissue microarrays was used to characterise α1b, α2c and ß(2)2 adrenoceptor protein expression in operable breast tumours. Associations with tumour-relevant biological markers and clinical outcome were statistically assessed. Strong α1b expression occurred in large high grade (P < 0.0001), HER2+ (P < 0.0001) or basal-like (CK5/6, P = 0.0005; CK14, P = 0.0001; EGFR, P = 0.003) cancers, showing increased proliferation (Mib1, P = 0.002), decreased apoptosis (Bcl2, P < 0.0001) and poor NPI membership (P = 0.001). α1b expression correlated with poor cancer-specific survival (LR = 7.628, P = 0.022) and tumour recurrence (LR = 6.128, P = 0.047). Strong α2c was over-expressed in high grade (P = 0.007), HER3+ (P = 0.002) and HER4+ (P < 0.0001) cancers with borderline increase in EGFR, p53 and MIB1 proteins, and inverse association with hormonal (PgR, P = 0.002) phenotype. In contrast, strong ß(2) expression occurred in small-size, luminal-like (ER+, P < 0.001) tumours of low grade (P < 0.001) and lymph node stage (P = 0.027) that showed poor prognosis when hormonal treatment was withheld. Adrenoceptors were not found to be independent predictors of clinical outcome. Alpha1b and α2c AR is over-expressed in basal-like breast tumours of poor prognosis. Strong ß(2) adrenoceptor expression is seen in patients with a luminal (ER+) tumour phenotype and good prognosis, due to benefits derived from hormonal therapy. These findings suggest a possible role for targeted therapy using adrenoceptor antagonists.

Tumour reactions to hypoxia.

Fast growing solid tumors generally lack an inner organisation, which causes the problem of a sufficient nutrient of each part of the tumor that then happens only by diffusion. The low oxygen supply leads to the activation of hypoxia-inducible factors, which regulate a plethora of genes. The reaction of tumor cells to hypoxia can be divided into two parts: On the one hand, there are signal substances, predominantly growth factors and cytokines, which provoke the vascularisation (angiogenesis), lymph vessel development (lymphangiogenesis), and the innervation (neoneurogenesis) of tumors and thus connect the tumor to structures of the environment. On the other hand, genes for intracellular proteins and receptors are regulated, which lead to changes of the tumor cell functions. Best characterized is the metabolic shift, a high anaerobic glycolytic activity and simultaneously a reduction of respiration. Furthermore, proliferation, dedifferentiation, resistance to apoptosis, and the metastatic potential are affected. With regard to the latter, we herein show that the migratory activity and velocity of PC-3 human prostate carcinoma cells significantly increases under oxygen-deprivation, which might be an explanation for the increasing number of experimental and clinical hints, that an anti-angiogenic therapy can promote the metastasis formation.

Surface molecules regulating rolling and adhesion to endothelium of neutrophil granulocytes and MDA-MB-468 breast carcinoma cells and their interaction.

The extravasation of leukocytes and tumor cells is a multi-step process with the involvement of various adhesion molecules depending on the three steps rolling, adhesion, and diapedesis. We have developed an in vitro model, by which we investigated the rolling and adhesion of neutrophil granulocytes and MDA-MB-468 human breast carcinoma cells to lung endothelial cells under physiological flow-conditions. We found that norepinephrine had an inhibitory function on the fMLP-promoted adhesion of neutrophil granulocytes due to a down-regulation of beta2-integrin. Furthermore, neutrophil granulocytes serve as linking cells for the interaction of the MDA-MB-468 cells with the endothelium, which are both beta2-integrin negative, but express the beta2-integrin ligand ICAM-1. In addition, we show here that N-cadherin is up-regulated on the endothelial cells and on neutrophil granulocytes in response to fMLP. This up-regulation resulted in a significant increase of adherent MDA-MB-468 cells, which are also N-cadherin positive.

N-cadherin engagement provides a dominant stop signal for the migration of MDA-MB-468 breast carcinoma cells.

The development of a primary tumor as such is not the main cause of death, but is rather the spreading of metastases, which causes over 90% of deaths in cancer patients. This largely depends on the ability of tumor cells to migrate away from the tumor and relocate at other areas of the body. Cell migration is known to be regulated by various extracellular signal substances such as neurotransmitters. However, before single tumor cells can start to invade into distant tissue, they have to dissociate from the primary tumor. This requires the disruption of cell-cell contacts, which are provided by a plethora of adhesion molecules like the family of cadherins. Using our well, established three-dimensional collagen-based cell migration assay, we show that engagement of N-cadherin results in a significant decrease of the spontaneous and the norepinephrine-induced migration of MDA-MB-468 breast carcinoma cells, which was due to an increase in the average break length. Moreover, this N-cadherin driven influence on the migratory activity is intracellularly integrated via multiple signaling pathways. Our results show that the impact of N-cadherin on the locomotion of MDA cells involves the activation of the adenylyl cyclase and the phosphatidylinositol-3-kinase (PI3K), but is independent of the protein kinase C (PKC) alpha. In summary, we provide evidence that the engagement of N-cadherin provides a stop signal for breast carcinoma cell migration, and accordingly the use of anti-N-cadherin antibodies or soluble ligands might be a tool to inhibit metastasis formation in E-cadherin negative but N-cadherin positive tumors.

DPPIV inhibitors extend GLP-2 mediated tumour promoting effects on intestinal cancer cells.

BACKGROUND: The glucagon-like peptides-1 and -2 (GLP-1 and -2) are co-secreted after food intake from intestinal L cells. Since both peptides are rapidly degraded by dipeptidyl peptidase-IV (DPPIV), research is focused on the development of DPPIV inhibitors or DPPIV resistant. AIMS: In this study we investigated, whether the inhibition of DPPIV activity and the resulting increased half-life of DPPIV substrates may influence cancer development and progression. METHODS: We examined proliferation and migratory activity of two human colon cancer cell lines (SW480, HT29) after stimulation with GLP-2 in combination with or without DPPIV inhibitors. RESULTS: Migratory activity was increased by 25% from 20% matrix induced activity to a maximum of 45% (100 nM GLP-2). In cells expressing CD26, migration was prolonged by addition of DPPIV inhibitors in a concentration dependent manner. After treatment with GLP-2 doubling time decreased from 2.4 to 1.5 days - and addition of DPPIV inhibitors enhanced the effect of GLP-2. CONCLUSIONS: The use of DPPIV inhibitors together with GLP-2 led to increased proliferation as well as elevated migratory activity. Therefore, the use of DPPIV inhibitors could increase the risk of promoting an already existing intestinal tumour and may support the potential of colon cancer cells to metastasize.

Myosin regulation in the migration of tumor cells and leukocytes within a three-dimensional collagen matrix.

The migration of cells is a complex regulatory process which results in the generation of motor forces through the reorganization of the cytoskeleton. Here we present a comparative study of the expression and involvement of myosin in the regulation of the physiological migration of leukocytes and the pathological migration of tumor cells. We show that the involvement of myosin in the migration is distinct in these two cell types. In leukocytes, the activity of non-muscle myosin II is essential for both the spontaneous (matrix-induced) migration and the migration induced by ligands to G protein-coupled receptors, i.e. chemokines and neurotransmitters. In contrast, spontaneous tumor cell migration is largely independent of non-muscle myosin II activity, whereas the norepinephrine-induced migration is completely inhibited by either direct inhibition of non-muscle myosin II or of the kinases phosphorylating the myosin light chain, namely ROCK or the calcium/calmodulin-dependent myosin light-chain kinase.

Effects of neurotransmitters on the chemokinesis and chemotaxis of MDA-MB-468 human breast carcinoma cells.

Most patients suffering from breast carcinoma do not die due to the primary tumor but from the development of metastases. Active migration of cancer cells is a prerequisite for development of these metastases. We used time-lapse videomicroscopy and computer-assisted cell tracking of MDA-MB-468 human breast carcinoma cells, which were incorporated into a three-dimensional collagen matrix, in order to analyze the migratory activity of these cells in response to different neurotransmitters. Our results show that met-enkephalin, substance P, bombesin, dopamine, and norepinephrine have a stimulatory effect on the migration of the breast cancer cells; moreover, these cells show positive chemotaxis towards norepinephrine as was analyzed by the directionality and persistence on a single-cell basis. Gamma-aminobutyric acid (GABA) however has an inhibitory effect. Endorphin and leu-enkephalin, as well as histamin and acetylcholine, had no influence on the migratory activity of the cells. In summary, we provide evidence for a strong regulatory involvement of neurotransmitters in the regulation of breast cancer cell migration, which might provide the basis for the use of the pharmacological agonists and antagonists for the chemopreventive inhibition of metastasis development.

A novel function for chemokines: downregulation of neutrophil migration.

Migration is a key function of stem cells during ontogenesis, of fibroblasts in wound healing and of immune cells in host defence. The signals that initiate migration are as important as signals that terminate migration, once the destination has been reached. We now show that formyl-methionyl-leucyl-phenylalanine (fMLP)-induced migration of neutrophils was inhibited by increasing concentrations of interleukin-8 (IL-8). IL-8 dose dependently increased the frequency and the duration of stop-periods, whereas the percentage of cells of a population that was locomotory active remained constant. The stop-signal delivered by IL-8 was intracellularly transduced by a dichotomic pathway: (i) the activation of the adenylyl cyclase leads to an increase of cytosolic cyclic adenosine monophosphate, which results in an activation of the sarcoplasmatic/endoplasmatic reticulum calcium ATPase pump and a calcium sequestration; (ii) the activation of the phospholipase Cbeta (PLCbeta) generates inositol-1,4,5-phosphate (IP3) and diacylglycerol (DAG), which results in IP3-mediated release of intracellularly stored calcium in the endoplasmatic reticulum and DAG-mediated activation of protein kinase C. Thus, we show for the first time that a chemokine, IL-8, in concert with fMLP, downregulates the neutrophil migration through the regulation of the intracellular calcium concentration via the adenylyl cyclase and the PLCbeta2.

Realtime visualization of tumor cell/endothelial cell interactions during transmigration across the endothelial barrier.

PURPOSE: In cancer the blood-borne spread of tumor cells leads to the formation of secondary tumors at distant loci whereby the extravasation of tumor cells is a prerequisite step during hematogenous metastasis. Here, we describe a novel in vitro realtime model which shows the complete sequence of the extravasation process. METHODS: We developed an in vitro system allowing us to monitor the sequence of extravasation events of tumor cell clusters across a monolayer of human umbilical cord endothelial cells (HUVEC). Fluorescence markers and laser scanning confocal microscopy were used to visualize the interactions between tumor cells and endothelium. RESULTS: Our model indicates that the extravasation of tumor cell clusters derived from the invasive human bladder carcinoma cell line T24 occurs in a relatively short time-frame up to 4 h after adhesion to the endothelium. We demonstrate that the vascular endothelium is irreversibly damaged at the site of tumor cell extravasation. CONCLUSION: Realtime laser scanning confocal microscopy leads to a better understanding of the complex and dynamic cell-to-cell and cell-to-matrix interactions during the extravasation process.

High PKC alpha and low E-cadherin expression contribute to high migratory activity of colon carcinoma cells.

The protein kinase C (PKC) is a family of serine/threonine kinases that are key regulatory enzymes involved in growth, differentiation, cytoskeletal reorganization, tumor promotion, and migration. We investigated the functional involvement of PKC isotypes and of E-cadherin in the regulation of the locomotion of six human colon-adenocarcinoma cell lines. The different levels of the PKC alpha and the E-cadherin expression have predictable implications in the spontaneous locomotory activity. With the use of PKC alpha--specific inhibitors (safingol, Go6976) as well as the PKC delta--specific inhibitor rottlerin, we showed that only PKC alpha plays a major role in the regulation of tumor cell migration. The results were verified by knocking out the translation of PKC isozymes with the use of an antisense oligonucleotide strategy. After stimulation with phorbol ester we observed a translocation and a colocalization of the activated PKC alpha at the plasma membrane to the surrounding extracellular matrix. Furthermore, we investigated the functional involvement of E-cadherin in the locomotion with the use of a blocking antibody. A high level of PKC alpha expression together with a low E-cadherin expression was strongly related to a high migratory activity of the colon carcinoma cells. This correlation was independent of the differentiation grade of the tumor cell lines.

Norepinephrine-induced migration of SW 480 colon carcinoma cells is inhibited by beta-blockers.

Beta-adrenoceptors are highly expressed on SW 480 colon carcinoma cells as was assessed by flow cytometry. We investigated the influence of norepinephrine on the migration of these cells using time-lapse videomicroscopy. Norepinephrine-treatment increased the locomotor activity within the population from 25% spontaneously locomoting cells to 65% locomoting cells. The beta1/2-blocker propranolol but not the beta1-blocker atenolol inhibited this increase. The intracellular signaling solely of norepinephrine-induced locomotion involved protein tyrosine kinase activity, whereas both spontaneous and norepinephrine-induced migration were reduced by inhibiting phospholipase Cgamma and protein kinase Calpha activity. In summary, norepinephrine-induced locomotion of SW 480 cells is beta2-adrenoceptor mediated and distinct from spontaneous locomotion concerning the PTK involvement.

Migration and metalloproteinases determine the invasive potential of mouse melanoma cells, but not melanin and telomerase.

The biological characteristic cell locomotion and invasion, melanin content, metalloproteinases and telomerase activity were studied in a parental mouse melanoma cell line B16 and two descendents B16BL6 and B16F10. The invasive potential of melanoma cells was assayed in a transwell cell culture chamber. Melanin content was determined by the absorbance value at 470 nm per 10(6) cells. Tumor cells migration within the 3-D collagen matrix was microscopically recorded with a time-lapse video recorder and analyzed by computer-assisted cell tracking. Gelatin zymography was adopted to assay the metalloproteinases secretion. A polymerase chain reaction-based telomeric repeat amplification protocol (TRAP) was used for measuring telomerase activity. The results demonstrated that B16BL6 and B16F10 cells were highly invasive compared to B16 cells, but the melanin content of B16F10 was very low. B16F10 and B16BL6 were hypermotile and secreted much more metalloproteinases than B16. No differences were observed in telomerase activity among the three melanoma cell lines. Invasion of mouse melanoma was closely correlated to tumor cell migration and secretion of metalloproteinases. Melanin content and telomerase activity were phenotypically not related to invasiveness in these three mouse melanoma cell lines.

T lymphocytes and neutrophil granulocytes differ in regulatory signaling and migratory dynamics with regard to spontaneous locomotion and chemotaxis.

Chemotactic migration of T lymphocytes and neutrophil granulocytes within a three-dimensional collagen matrix is distinct from spontaneous, matrix-induced migration concerning dynamic parameters and regulatory intracellular signaling. Both spontaneous T lymphocyte locomotion and stromal-cell-derived factor-1 (SDF-1)-induced chemotaxis-involved protein tyrosine kinase (PTK) activity, whereas only SDF-1-induced migration was protein kinase C (PKC) dependent. Spontaneous locomotion of neutrophil granulocytes was independent of PKC and PTK activity, but formyl-methionyl-leucyl-phenylalanine-induced migration involved PKC activity. In addition, the microtubule cytoskeleton was not changed after induction of chemotaxis in both cell types. T lymphocytes had a well-developed microtubule cytoskeleton with the microtubule organizing center located in the uropod, whereas neutrophil granulocytes revealed a clustered tubulin distribution at the leading edge of the migrating cell. Therefore, differences of the microtubule cytoskeleton might contribute to differences in locomotion between T lymphocytes and neutrophil granulocytes but not to differences between spontaneous locomotion and chemotaxis.

Locomotion of tumor cells: a molecular comparison to migrating pre- and postmitotic leukocytes.

Increasing evidence has shown that the molecular regulation of active cell migration of slow-moving cells, e.g., tumor cells and fibroblasts, is different from fast-moving leukocytes, e.g., T lymphocytes and neutrophil granulocytes. Slow-moving cells develop focal adhesions as a crucial regulatory element during migration. These focal adhesions connect the extracellular matrix to the intracellular actin- and tubulin-cytoskeleton via integrins and enzymatically active proteins. Beside matrix-binding integrins, ligands of receptor tyrosine kinases and heterotrimeric G protein-coupled serpentine receptors initiate migration of slow-moving cells. Focal adhesions are not found in T lymphocytes and neutrophil granulocytes moving within three-dimensional matrices. In T lymphocytes, the T cell receptor is supposed to have a key regulatory function not only in antigen recognition, cell activation, and proliferation but also in cell migration. Regulatory molecules as well as the cytoskeleton are connected to the T cell receptor. The T cell receptor functionally combines elements of receptor tyrosine kinase signaling and of focal adhesions. In neutrophil granulocytes no multi-protein complexes regulating migration have been identified so far. Most potent activators of migration of neutrophil granulocytes, as those of T lymphocytes, are chemokines binding to heterotrimeric G protein-coupled serpentine receptors.

Lessons from tumor and immunocompetent cells. The quantitative engagement of ligand-receptor interactions modulates stop-and-go behavior as well as proliferation.

The four main cell functions, proliferation, apoptosis, differentiation and migration, are tightly regulated by external signals that initiate intracellular signal transduction pathways and determine the cellular behaviour. The concentration and composition of such external signals are at least important for the decision of cells as to which function has to be executed. Interleukin-8 is a well known inducing signal for neutrophil granulocyte migration, while the epidermal growth factor is an inducing signal for breast carcinoma cell migration. Depending on the concentrations of interleukin-8, the neutrophil granulocytes are capable of migration. However, at high concentration of interleukin-8 the migratory activity of each single cell is reduced, indicating that high concentrations of the chemokine inhibit migration and promote the performance of other cell functions. Concerning breast carcinoma cells, the epidermal growth factor is not only an inducer of migration but also an inhibitor of proliferation. These two examples provide evidence for a dose dependent action of external signals for several cell functions in parallel. This versatility of the effects of one ligand might be based on several intracellular signal transduction pathways that are turned on. For the dose-dependent differences of the effect of interleukin-8 we propose a two wheel model of an inositolphosphate-mediated, ATP-independent release of calcium from intracellular stores and a cyclic AMP-mediated, ATP-dependent uptake of calcium into the endoplasmatic reticulum.

Lessons from tumor and immunocompetent cells. The quantitative engagement of ligand-receptor interactions modulates stop-and-go behavior as well as proliferation

The four main cell functions, proliferation, apoptosis, differentiation and migration, are tightly regulated by external signals that initiate intracellular signal transduction pathways and determine the cellular behaviour. The concentration and composition of such external signals are at least important for the decision of cells as to which function has to be executed. Interleukin-8 is a well known inducing signal for neutrophil granulocyte migration, while the epidermal growth factor is an inducing signal for breast carcinoma cell migration. Depending on the concentrations of interleukin-8, the neutrophil granulocytes are capable of migration. However, at high concentration of interleukin-8 the migratory activity of each single cell is reduced, indicating that high concentrations of the chemokine inhibit migration and promote the performance of other cell functions. Concerning breast carcinoma cells, the epidermal growth factor is not only an inducer of migration but also an inhibitor of proliferation. These two examples provide evidence for a dose dependent action of external signals for several cell functions in parallel. This versatility of the effects of one ligand might be based on several intracellular signal transduction pathways that are turned on. For the dose-dependent differences of the effect of interleukin-8 we propose a two wheel model of an inositolphosphate-mediated, ATP-independent release of calcium from intracellular stores and a cyclic AMP-mediated, ATP-dependent uptake of calcium into the endoplasmatic reticulum.

Lessons from tumor and immunocompetent cells. The quantitative engagement of ligand-receptor interactions modulates stop-and-go behavior as well as proliferation.

The four main cell functions, proliferation, apoptosis, differentiation and migration, are tightly regulated by external signals that initiate intracellular signal transduction pathways and determine the cellular behaviour. The concentration and composition of such external signals are at least important for the decision of cells as to which function has to be executed. Interleukin-8 is a well known inducing signal for neutrophil granulocyte migration, while the epidermal growth factor is an inducing signal for breast carcinoma cell migration. Depending on the concentrations of interleukin-8, the neutrophil granulocytes are capable of migration. However, at high concentration of interleukin-8 the migratory activity of each single cell is reduced, indicating that high concentrations of the chemokine inhibit migration and promote the performance of other cell functions. Concerning breast carcinoma cells, the epidermal growth factor is not only an inducer of migration but also an inhibitor of proliferation. These two examples provide evidence for a dose dependent action of external signals for several cell functions in parallel. This versatility of the effects of one ligand might be based on several intracellular signal transduction pathways that are turned on. For the dose-dependent differences of the effect of interleukin-8 we propose a two wheel model of an inositolphosphate-mediated, ATP-independent release of calcium from intracellular stores and a cyclic AMP-mediated, ATP-dependent uptake of calcium into the endoplasmatic reticulum.

CD4+ T lymphocytes migrating in three-dimensional collagen lattices lack focal adhesions and utilize beta1 integrin-independent strategies for polarization, interaction with collagen fibers and locomotion.

Cell migration may depend on integrin-mediated adhesion to and deadhesion from extracellular matrix ligands. This concept, however, has not yet been confirmed for T lymphocytes migrating in three-dimensional extracellular matrices. We investigated receptor involvement in T cell migration combining a three-dimensional collagen matrix model with time-lapse videomicroscopy, computer-assisted cell tracking and confocal microscopy. In collagen lattices, the migration of CD4+ T cells (1) involved interactions with collagen fibers at the leading edge and uropod likewise, (2) occurred independently of the co-clustering of beta1, beta2, or beta3 integrins with F-actin, focal adhesion kinase, and phosphotyrosine at interactions with collagen fibers, (3) was counteracted by high-affinity beta1 integrin binding induced by antibody TS2/16; however, (4) the migration could not be blocked by a combination of adhesion-perturbing anti-beta1, -beta2, -beta3, and alpha v integrin antibodies. Integrin blocking neither affected cell polarization, interaction with fibers, beta1 integrin distribution, migration velocity, path structure, nor the number of locomoting cells in spontaneously migrating or concanavalin A-activated cells. Hence, T lymphocytes migrating in three-dimensional collagen matrices may utilize highly transient interactions with collagen fibers of low adhesivity, thereby differing from focal adhesion-dependent migration strategies employed by other cells.

Differential requirement of protein tyrosine kinases and protein kinase C in the regulation of T cell locomotion in three-dimensional collagen matrices.

Locomotion of T lymphocytes within three-dimensional collagen matrices is regulated via different signaling states of the cells. Purified human CD4+ and CD8+ T cells developed a spontaneously locomoting subpopulation of about 25% of the whole population immediately after incorporation into a three-dimensional collagen matrix analyzed by time-lapse videomicroscopy. This spontaneous locomotion was accompanied by enhanced tyrosine phosphorylation of the focal adhesion kinase (FAK). Inhibition of protein tyrosine kinase (PTK) activity using genistein significantly reduced the spontaneous locomotory activity. This reduction was overcome by subsequent activation of protein kinase C (PKC) using PMA, which led to a persistent increase of locomotory activity to more than 60% of the cells. Thus, the PKC-driven type of locomotion was independent of PTK activity, whereas spontaneous locomotion was not altered by inhibition of PKC activity using calphostin C or inhibition of the serine/ threonine phosphatases pp1 and pp2A using okadaic acid. We presume that PTK activity, especially tyrosine phosphorylation of FAK, is decisively involved in the regulation of spontaneous T lymphocyte locomotion, which is independent of PKC activity. In contrast, PKC-driven locomotion is independent of tyrosine phosphorylation events, indicating that T lymphocyte locomotion is regulated by more than one signal transduction pathway. Furthermore, confocal microscopy analysis of phosphotyrosine residues, FAK, and PKC revealed an exclusive cellular distribution of these components, suggesting a regulation of T lymphocyte locomotion different from migration models developed for other cell types, which refer to a colocalization of FAK and PKC in focal adhesions.

Purification, structural analysis, and function of natural ATAC, a cytokine secreted by CD8(+) T cells.

Recently, we identified a novel putative human cytokine expressed by activated CD8(+) T cells, which was designated ATAC (activation-induced, T cell-derived, and chemokine-related; the same molecule has been identified independently as lymphotactin and single cysteine motif-1). In this report, we provide evidence that ATAC is a secreted 93-amino acid protein that is generated from its precursor by proteolytic cleavage between Gly21 and Val22. An estimated 60% of ATAC (Val22-Gly114) is secreted as an unmodified protein with a molecular mass of 10,271.72 Da (apparent molecular mass of 12 kDa in SDS-polyacrylamide gel electrophoresis) and in which Cys32 and Cys69 are linked by a disulfide bridge. Unmodified ATAC is a cationic protein with a pI of 11.35 and is capable of binding to heparin. Some 40% of ATAC is O-glycosylated within 25 min of synthesis, giving rise to the appearance of a homogeneous 15-kDa (minor fraction) and a heterogeneous, terminally sialylated 17-19-kDa (major fraction) protein species in SDS-polyacrylamide gel electrophoresis. The secretion of all ATAC protein variants is completed within 30-40 min of synthesis. In terms of function, various ATAC protein forms were consistently ineffective in chemotaxis assays. In contrast, both purified natural ATAC and a chemically synthesized aglycosyl analog induced locomotion (chemokinesis) in purified CD4(+) and CD8(+) T cell populations at 400 ng/ml.