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The cell cycle is strictly programmed with control mechanisms that dictate order in cell cycle progression to ensure faithful DNA replication, whose deviance may lead to cancer. Checkpoint control at the G1/S, S/G2 and G2/M portals have been defined but no statutory time-programmed control for securing orderly transition through S phase has so far been identified. Here we report that in normal cells DNA synthesis is controlled by a checkpoint sited within the early part of S phase, enforced by the βGBP cytokine an antiproliferative molecule otherwise known for its oncosuppressor properties that normal cells constitutively produce for self-regulation. Suppression of active Ras and active MAPK, block of cyclin A gene expression and suppression of CDK2-cyclin A activity are events which while specific to the control of a cell cycle phase in normal cells are part of the apoptotic network in cancer cells.

Phosphoinositide 3-kinase (PI3K)-activated signalling has a critical role in the evolution of aggressive tumourigenesis and is therefore a prime target for anticancer therapy. Previously we have shown that the β galactoside binding protein (βGBP) cytokine, an antiproliferative molecule, induces functional inhibition of class 1A and class 1B PI3K. Here, we have investigated whether, by targeting PI3K, βGBP has therapeutic efficacy in aggressive breast cancer cells where strong mitogenic input is fuelled by overexpression of the ErbB2 (also known as HER/neu, for human epidermal growth factor receptor 2) oncoprotein receptor and have used immortalised ductal cells and non-aggressive mammary cancer cells, which express ErbB2 at low levels, as controls. Aggressive BT474 and SKBR3 cancer cells where ErbB2 is overexpressed, MCF10A immortalised ductal cells and non-invasive MCF-7 cancer cells which express low levels of ErbB2, both in their naive state and when forced to mimic aggressive behaviour, were used. Class IA PI3K was immunoprecipitated and the conversion of phosphatidylinositol (4,5)-biphosphate (PIP2) to phosphatidylinositol (3,4,5)-trisphosphate (PIP3) assessed by ELISA. The consequences of PI3K inhibition by βGBP were analysed at proliferation level, by extracellular signal-regulated kinase (ERK) activation, by akt gene expression and by apoptosis. Apoptosis was documented by changes in mitochondrial membrane potential, alteration of the plasma membrane, caspase 3 activation and DNA fragmentation. Phosphorylated and total ERK were measured by Western blot analysis and akt mRNA levels by Northern blot analysis. The results obtained with the BT474 and SKBR3 cells were validated in the MCF10A ductal cells and in non-invasive MCF-7 breast cancer cells forced into mimicking the in vitrobehaviour of the BT474 and SKBR3 cells. In aggressive breast cancer cells, where mitogenic signalling is enforced by the ErbB2 oncoprotein receptor, functional inhibition of the catalytic activity of PI3K by the βGBP cytokine and loss of akt mRNA results in apoptotic death. A functional correlation between ERK and the kt gene was also found. The relationship between ERK, akt mRNA, PI3K and cell vulnerability to βGBP challenge was sustained both in mammary ductal cells forced to mimic an aggressive behaviour and in non-aggressive breast cancer cells undergoing an enforced shift into an aggressive phenotype. βGBP, a newly discovered physiological inhibitor of PI3K, is a selective and potent inducer of apoptosis in aggressive breast cancer cells. Due to its physiological nature, which carries no chemotherapeutic disadvantages, βGBP has the potential to be safely tested in clinical trials.

The cell cycle is negatively regulated by diverse molecular events which originate in part from the interaction of secreted proteins with specific cell surface receptors. By exerting negative control on cell proliferation, these factors can help maintain cell number balance both through growth restraints and the induction of apoptosis and may thus contribute to prevent or control tumourigenesis. Here we report that βGBP, a negative growth factor which controls transition from S phase into G2, causes an S/G2 growth arrest in both normal and leukaemic T cells. However, in leukaemic T cells but not in normal T lymphocytes, growth arrest is followed by apoptosis. Analysis of possible mechanisms of induction of apoptosis does not support Fas and Fas L as having a main role but points instead to Bcl-2 and Bax. The induction of apoptosis in leukaemic T cells is characterised by the decrease of Bcl-2 and consequent predominance of Bax. By contrast, in the normal T cells, which do not enter apoptosis, the quantitative relationship of Bcl-2 to Bax remains unchanged. The ability of βGBP to selectively induce apoptosis in leukaemic cells suggests that βGBP may play a role in cancer surveillance and that its use has potential therapeutic implications. J Cell Physiol 178:102–108, 1999. © 1999 Wiley-Liss, Inc.

CONSIDERABLE evidence derived from the study of animal cells cultured in vitroimplicates the cell surface in the regulation of cell division and in the expression of the phenotypic state of the cell. Cell surface is a broad term used to indicate the lipid bilayer of the cytoplasmic membrane containing intrinsic (stably integrated) and peripheral (easily removable) protein constituents plus carbohydrate complexes1–3. There are indications that some of the intrinsic constituents are of fundamental importance in certain physiological processes such as ion transport and transport of metabolic precursors4–8. On the other hand, it is not known whether components that are more loosely bound to the cell surface play any specific role in cell physiology because no cellular function has so far been examined in relation to such components. We report here that determination of cell shape is affected by a cell surface protein component easily detachable from the cell exterior.

βGBP is a novel physiological negative growth regulator of the cell and a cytostatic factor.It is secreted by cells, and bybinding with high affinity to specific cell surface receptors. In normal cell surface receptors. In normal cells, βGBP physiologically controls transition from G0 to G1 and passage from late S phase to G2 by modulating signalling cascades activated by tyrosine Kinase receptors and by affecting transcription events.As a cytostatic Factor βGBP has a marked growth inhibitory effect on a variety of tumours including leukaemias where growth arrest is followed by the activation of apoptotic pathways and cell death.

We have used mRNA differential display PCR to search for genes induced in activated T cells and have found the LGALS1 (lectin, galactoside-binding, soluble) gene to be strongly up-regulated in effector T cells. The protein coded by the LGALS1 gene is a β-galactoside-binding protein (βGBP), which is released by cells as a monomeric negative growth factor but which can also associate into homodimers (galectin-1) with lectin properties. Northern blot analysis revealed that ex vivo isolated CD8⁺ effector T cells induced by a viral infection expressed high amounts of LGALS1 mRNA, whereas LGALS1 expression was almost absent in resting CD8⁺ T cells. LGALS1 expression could be induced in CD4⁺ and CD8⁺ T cells upon activation with the cognate peptide antigen and high levels of LGALS1 expression were found in concanavalin A-activated T cells but not in lipopolysaccharide-activated B cells. Gel filtration and Western blot analysis revealed that only monomeric βGBP was released by activated CD8⁺ T cells and in vitro experiments further showed that recombinant βGBP was able to inhibit antigen-induced proliferation of naive and antigen-experienced CD8⁺ T cells. Thus, these data indicate a role of βGBP as an autocrine negative growth factor for CD8⁺ T cells.

Increase in cell numbers is inhibited by adding mecillinam to vegetative cultures ofMyxococcus xanthus. However, both cell length and volume continue to increase in the presence of the antibiotic. Different size classes of cells increase in proportion to their initial size. Incorporation ofmeso-diamino[14C]pimelic acid into peptidoglycan and [3H]uridine into RNA is not immediately affected by mecillinam. It is suggested that mecillinam inhibits the formation of new sites of wall synthesis, and these are necessary for cell division to occur.

The structural gene for β-galactoside-binding protein (Lgals-1), a cell growth regulatory molecule and cystostatic factor, is assigned to the E-region of mouse chromosome 15 and to the region q12-q13.1 of human chromosome 22. The evolutionary conservation of these two regions has been previously suggested from comparative mapping of several loci. These include the murine SIS oncogene (Pdgfb) and its human homolog, the platelet-derived growth factor-β polypeptide, PDGFB. The findings presented here extend the genetic homology of the two regions. Copyright 1993, 1999 Academic Press

βGBP is a novel physiological negative growth regulator of the cell and a cytostatic factor.It is secreted by cells, and bybinding with high affinity to specific cell surface receptors. In normal cell surface receptors. In normal cells, βGBP physiologically controls transition from G0 to G1 and passage from late S phase to G2 by modulating signalling cascades activated by tyrosine Kinase receptors and by affecting transcription events.As a cytostatic Factor βGBP has a marked growth inhibitory effect on a variety of tumours including leukaemias where growth arrest is followed by the activation of apoptotic pathways and cell death.