Background Immunor (IM28), an analog of dehydroepiandrosterone (DHEA), inhibits human immunodeficiency virus type-1 (HIV-1) by inhibiting reverse transcriptase. We assessed the ability of IM28 to inhibit the cell-cell fusion mediated by HIV envelope glycoprotein in an in vitro system. For this purpose, we co-cultured TF228.1.16, a T-cell line expressing stably HIV-1 glycoprotein envelopes, with an equal number of 293/CD4+, another T cell line expressing CD4, and with the SupT1 cell line with or without IM28.
Background
Immunor (IM28), an analog of dehydroepiandrosterone (DHEA), inhibits human immunodeficiency virus type-1 (HIV-1) by inhibiting reverse transcriptase. We assessed the ability of IM28 to inhibit the cell-cell fusion mediated by HIV envelope glycoprotein in an in vitro system. For this purpose, we co-cultured TF228.1.16, a T-cell line expressing stably HIV-1 glycoprotein envelopes, with an equal number of 293/CD4+, another T cell line expressing CD4, and with the SupT1 cell line with or without IM28.Results In the absence of IM28, TF228.1.16 fused with 293/CD4+, inducing numerous large syncytia. Syncytia appeared more rapidly when TF228.1.16 was co-cultured with SupT1 cells than when it was co-cultured with the 293/CD4+ cell line. IM28 (1.6 – 45 μg/ml) completely inhibits cell-cell fusion. IM28 also prevented the development of new syncytia in infected cells and protected naive SupT1 cells from HIV-1 infection. Evaluation of 50% inhibitory dose (IC50) of IM28 revealed a decrease in HIV-1 replication with an IC50 of 22 mM and 50% cytotoxicity dose (CC50) as determined on MT2 cells was 75 mM giving a selectivity index of 3.4
Results
In the absence of IM28, TF228.1.16 fused with 293/CD4+, inducing numerous large syncytia. Syncytia appeared more rapidly when TF228.1.16 was co-cultured with SupT1 cells than when it was co-cultured with the 293/CD4+ cell line. IM28 (1.6 – 45 μg/ml) completely inhibits cell-cell fusion. IM28 also prevented the development of new syncytia in infected cells and protected naive SupT1 cells from HIV-1 infection. Evaluation of 50% inhibitory dose (IC50) of IM28 revealed a decrease in HIV-1 replication with an IC50 of 22 mM and 50% cytotoxicity dose (CC50) as determined on MT2 cells was 75 mM giving a selectivity index of 3.4Conclusions These findings suggest that IM28 exerts an inhibitory action on the env proteins that mediate cell-cell fusion between infected and healthy cells. They also suggest that IM28 interferes with biochemical processes to stop the progression of existing syncytia. This property may lead to the development of a new class of therapeutic drug.
Conclusions
These findings suggest that IM28 exerts an inhibitory action on the env proteins that mediate cell-cell fusion between infected and healthy cells. They also suggest that IM28 interferes with biochemical processes to stop the progression of existing syncytia. This property may lead to the development of a new class of therapeutic drug.

Background The human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein is composed of two subunits: a surface glycoprotein (gp120) and a trans-membrane glycoprotein (gp41). These two subunits interact with each other in a non covalent manner. Gp120 is critical for attachment to host cell CD4 receptors, whereas gp41 contains the fusion sequence. HIV and simian immunodeficiency virus (SIV) require a co-receptor in addition to CD4 for entry into cells. Primary HIV can use a broad range of co-receptor molecules, including CCR1, CCR2b, CCR3, CCR4 and CXCR4 [ 1 - 3 ]. However, expression of a co-receptor together with CD4 on some cell types does not confer susceptibility to infection [ 1 ]. Not all human cell types that express an appropriate co-receptor support virus replication, indicating that other factors that affect viral tropism are present. HIV-1 viral entry is inhibited in the presence of the ligands to these chemokine receptors. RANTES, MIP-1α and MIP-1β, all of which are ligands for CCR5, inhibit macrophage-tropic isolates, whereas SDF-1, the specific ligand for CXCR4, inhibits entry by T-cell-tropic isolates [ 4 - 6 ]. The ability of HIV-1 envelope glycoproteins to induce cell-cell fusion is an interesting property because molecules that inhibit the fusion process are possible antiviral drugs and may lead to the identification of important functional regions either on the viral glycoprotein or on cell membranes. A hydrophobic, 25-amino acid, conserved segment located at the N-terminus of gp41 and gp120/41 has been shown to be involved in the fusion reaction between the viral envelope and the host cell plasma membrane [ 7 , 8 ]. There is evidence suggesting that this sequence is also involved in the cytopathic process underlying HIV-1 infection of target cells [ 9 , 10 ]. Exposure of this hydrophobic peptide to the aqueous environment in the vicinity of the target cell initially depends on gp120/41 function [ 11 ]. This protein is activated after interacting with primary receptor CD4. This activation requires the presence of human co-factors [ 12 , 13 ]. According to this model, further interaction of the fusion peptide to bind membrane lipid with the cell membrane depends mainly on the ability of the peptide to bind membrane lipid components. Hence, drugs that are able to interfere with membrane proteins became relevant for the therapy of HIV, even though it is still important to inhibit virus replication. We have previously shown that IM28 can inhibit HIV-1 reverse transcriptase activity [ 14 ]. Here, we assessed its capacity to inhibit the fusion of HIV-1-infected cells to naive cells. We found that IM28 was able to inhibit cell-cell fusion in an in vitro system. We showed that IM28 significantly blocks HIV-1 glycoprotein-mediated cell-cell fusion.
Background
The human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein is composed of two subunits: a surface glycoprotein (gp120) and a trans-membrane glycoprotein (gp41). These two subunits interact with each other in a non covalent manner. Gp120 is critical for attachment to host cell CD4 receptors, whereas gp41 contains the fusion sequence. HIV and simian immunodeficiency virus (SIV) require a co-receptor in addition to CD4 for entry into cells. Primary HIV can use a broad range of co-receptor molecules, including CCR1, CCR2b, CCR3, CCR4 and CXCR4 [ 1 - 3 ]. However, expression of a co-receptor together with CD4 on some cell types does not confer susceptibility to infection [ 1 ]. Not all human cell types that express an appropriate co-receptor support virus replication, indicating that other factors that affect viral tropism are present. HIV-1 viral entry is inhibited in the presence of the ligands to these chemokine receptors. RANTES, MIP-1α and MIP-1β, all of which are ligands for CCR5, inhibit macrophage-tropic isolates, whereas SDF-1, the specific ligand for CXCR4, inhibits entry by T-cell-tropic isolates [ 4 - 6 ]. The ability of HIV-1 envelope glycoproteins to induce cell-cell fusion is an interesting property because molecules that inhibit the fusion process are possible antiviral drugs and may lead to the identification of important functional regions either on the viral glycoprotein or on cell membranes. A hydrophobic, 25-amino acid, conserved segment located at the N-terminus of gp41 and gp120/41 has been shown to be involved in the fusion reaction between the viral envelope and the host cell plasma membrane [ 7 , 8 ]. There is evidence suggesting that this sequence is also involved in the cytopathic process underlying HIV-1 infection of target cells [ 9 , 10 ]. Exposure of this hydrophobic peptide to the aqueous environment in the vicinity of the target cell initially depends on gp120/41 function [ 11 ]. This protein is activated after interacting with primary receptor CD4. This activation requires the presence of human co-factors [ 12 , 13 ]. According to this model, further interaction of the fusion peptide to bind membrane lipid with the cell membrane depends mainly on the ability of the peptide to bind membrane lipid components. Hence, drugs that are able to interfere with membrane proteins became relevant for the therapy of HIV, even though it is still important to inhibit virus replication. We have previously shown that IM28 can inhibit HIV-1 reverse transcriptase activity [ 14 ]. Here, we assessed its capacity to inhibit the fusion of HIV-1-infected cells to naive cells. We found that IM28 was able to inhibit cell-cell fusion in an in vitro system. We showed that IM28 significantly blocks HIV-1 glycoprotein-mediated cell-cell fusion.Results We determined the concentrations of various drugs required to inhibit and to partially inhibit the fusion of TF228.1.16 and 293/CD4+ (Table  1 ). All these drugs decreased the percentage of surface covered by syncytia. The concentration of IM28 (6.43 μg/ml) that inhibited the formation of syncytia was similar to that of DXSF 500 000 (3.52 μg/ml) (Table  1 ). There were no statistical differences between the inhibitory concentrations of any of the drugs tested and IM28. To confirm these observations, we used SupT1 cells because fusion takes place more rapidly in these cells. These cells were mixed with TF22.1.16 cells in the presence or absence of dexamethasone or IM28 and fusion was examined by light microscopy after various periods of co-cultivation. In the absence of dexamethasone or IM28, TF228.1.16 cells fused with SupT1 cells, forming aggregates (Figure  1a ). Infected cells were spindle-shaped with large syncytia after overnight culture (Figure  1b ). Table 1 Effect of drugs on fusion of TF228.1.16 cells to 293/CD4+ cells Treatment Effect § None F F F IM28 F (0.60) P (1.83) I (6.43) Dexamethasone F (0.48) P (1.67) I (5.20) Con A F (0.09) P (0.22) I (0.79) Heparin F (2.70) P (7.00) I (22.0) Suramin F (1.57) P (3.90) I (15.0) Dextran Sulfate 10,000 F (0.02) P (0.06) I (0.20) Dextran Sulfate 500,000 F (0.37) P (1.15) I (3.52) § F = 50–60% of the surface is covered by syncytia; P = partial inhibition of fusion: < 10% of the surface is covered by syncytia; I = inhibition of syncytia formation. TF228.1.16 cells were mixed with 293/CD4 +  cells (1:1 cell ratio) and transferred to a 24-well plate (10 5  cells per well in 200 μl of culture medium). TF228.1.16 cells and 293/CD4 +  cells were incubated in the presence or absence of the drug (the final concentration in μg/ml is indicated in parenthesis) for 18 h. Following co-culture, three random fields of cells were photographed (not shown) and percentage fusion was determined as previously described [10]. Figure 1 Photomicrograph of SupT1 cells co-cultivated with TF 228.1.16 cells. Cell forming syncytia are aggregated (A). In the presence of dexamethasone (B) cells are mainly exploded vs. in the presence of IM28. In the presence of dexamethasone (Figure  2 ) or IM28 (Figure  3 ), the fusion of TF228.1.16 and SupT1 cells was completely inhibited in a dose-dependent manner. Indeed, in the presence of 0.5 μg/ml dexamethasone or IM28, time of incubation had no effect on syncytia formation. This concentration of dexamethasone or IM28 did not result in the lysis of existing syncytia but stopped the fusion reaction and the appearance of new syncytia (Figure  3 ). The time of incubation did not affect the inhibition of syncytia in the presence of dexamethasone, but did have an effect for 0.5 μg/ml IM28. In addition, the highest concentration (> 0.5 μg/ml) of both drugs completely inhibited syncytia formation. At this concentration of dexamethasone, the inhibition of syncytia was accompanied by cell death bursting (Figure  4 ), whereas the same concentration of IM28 did not lead to the burst (Figure  4 ). Figure 2 Effect of dexamethasone on fusion of TF228.1.16 cells with SupT1 cells. TF228.1.16 cells were mixed with SupT11 cells (1:1 cell ratio) and transferred to a 24-well plate (105 cells per well in 200 ml of cultured medium). After 24 h of co-culture in the presence or absence of dexamethasone (10 mg/ml), three random fields of cells were photographed and the percentage fusion was determined as described in Table 1.    Figure 3 Effect of IM28 on fusion of TF228.1.16 cells with SupT1 cells. TF228.1.16 cells were mixed with SupT11 cells (1:1 cell ratio) and transferred to a 24-well plate (10 5  cells per well in 200 μl of cultured medium). After 24 h of co-culture in the presence or absence of corticosteroids (dexamethasone or IM28) (10 μg/ml), three random fields of cells were photographed and the percentage fusion was determined as described in Table 1. Figure 4 Effect of IM28 and dexamethasone on SupT1 cells co-cultured with TF228.1.16. Zoom of negative photomicrograph of SupT1 cultures co-cultivated with TF 228.1.16 cells (A) in the presence of dexamethasone (B) and IM28. Note the evident syncytia in (A) with an apparent slender shape of infected cells. Cells treated with dexamethasone were atrophic and sometimes exploded whereas cells incubated with IM28 were round.    To further characterize the biological effect of the drug, the 50% inhibitory dose (IC50) and the cytotoxic dose (CC50) of IM28 were evaluated and the selectivity index which is the CC50/IC50 ratio was determined. The decrease in HIV-1 replication was obtained with an IC50 of 22 mM and the CC50 as determined on MT2 cells was 75 mM giving a selectivity index of 3.4.
Results
We determined the concentrations of various drugs required to inhibit and to partially inhibit the fusion of TF228.1.16 and 293/CD4+ (Table  1 ). All these drugs decreased the percentage of surface covered by syncytia. The concentration of IM28 (6.43 μg/ml) that inhibited the formation of syncytia was similar to that of DXSF 500 000 (3.52 μg/ml) (Table  1 ). There were no statistical differences between the inhibitory concentrations of any of the drugs tested and IM28. To confirm these observations, we used SupT1 cells because fusion takes place more rapidly in these cells. These cells were mixed with TF22.1.16 cells in the presence or absence of dexamethasone or IM28 and fusion was examined by light microscopy after various periods of co-cultivation. In the absence of dexamethasone or IM28, TF228.1.16 cells fused with SupT1 cells, forming aggregates (Figure  1a ). Infected cells were spindle-shaped with large syncytia after overnight culture (Figure  1b ).Effect of drugs on fusion of TF228.1.16 cells to 293/CD4+ cells§ F = 50–60% of the surface is covered by syncytia; P = partial inhibition of fusion: < 10% of the surface is covered by syncytia; I = inhibition of syncytia formation.TF228.1.16 cells were mixed with 293/CD4 +  cells (1:1 cell ratio) and transferred to a 24-well plate (10 5  cells per well in 200 μl of culture medium). TF228.1.16 cells and 293/CD4 +  cells were incubated in the presence or absence of the drug (the final concentration in μg/ml is indicated in parenthesis) for 18 h. Following co-culture, three random fields of cells were photographed (not shown) and percentage fusion was determined as previously described [10].In the presence of dexamethasone (Figure  2 ) or IM28 (Figure  3 ), the fusion of TF228.1.16 and SupT1 cells was completely inhibited in a dose-dependent manner. Indeed, in the presence of 0.5 μg/ml dexamethasone or IM28, time of incubation had no effect on syncytia formation. This concentration of dexamethasone or IM28 did not result in the lysis of existing syncytia but stopped the fusion reaction and the appearance of new syncytia (Figure  3 ). The time of incubation did not affect the inhibition of syncytia in the presence of dexamethasone, but did have an effect for 0.5 μg/ml IM28. In addition, the highest concentration (> 0.5 μg/ml) of both drugs completely inhibited syncytia formation. At this concentration of dexamethasone, the inhibition of syncytia was accompanied by cell death bursting (Figure  4 ), whereas the same concentration of IM28 did not lead to the burst (Figure  4 ).To further characterize the biological effect of the drug, the 50% inhibitory dose (IC50) and the cytotoxic dose (CC50) of IM28 were evaluated and the selectivity index which is the CC50/IC50 ratio was determined. The decrease in HIV-1 replication was obtained with an IC50 of 22 mM and the CC50 as determined on MT2 cells was 75 mM giving a selectivity index of 3.4.Discussion IM28 is a potent new derivative of DHEA that can stop the replication of HIV-1 by inhibiting its reverse transcriptase activity [ 14 ]. Here, we show that IM28 can also prevent and inhibit the fusion of infected cells (TF228.1.16 cells) to naïve cells including 293/CD4+ cells, which are stably transfected with human CD4 and highly susceptible to HIV-1 infection, and SupT1 cells [ 15 , 16 ]. The fusion of 293/CD4+ cells with TF228.1.16 cells was completely inhibited by a lower dose of IM28 than was the fusion of SupT1 cells with TF228.1.16 cells (data not shown). The fusion of TF228.1.16 cells to H4CD4+ (CD4 positive glial cell line) cells obtained by transfection of human neuroglioma cells [ 17 ] is also inhibited by IM28 (not shown). Therefore, IM28 and dexamethasone may inhibit cell-cell fusion and recombination-induced fusion mediated by the HIV env protein. Although the precise site at which IM28 acts to inhibit cell-cell fusion remains unknown, our results suggest that IM28 fights the HIV-1 virus at a new site. It is possible that this drug interacts with phospholipase A2 (PLA2), which plays an important role in the entry of HIV virus in the host cell [ 18 , 19 ]. Indeed, dexamethasone, a glucocorticoid, can inhibit the HIV-1, HIV-2 and SIVmac251 envelope glycoproteins and activate PLA2. PLA2 is activated when the envelope glycoprotein interacts with CD4. Due to its local membrane-destabilizing effect, PLA2 may play an important role in preparing the cell membrane for fusion with the viral particle. Activated PLA2 hydrolyzes membrane phospholipids in the sn-2 position, producing arachidonic acid and lysophospholipids [ 20 ]. These biochemical events also have downstream effects; the membrane is destabilized locally [ 21 , 22 ], and arachidonic acid and lysophospholipids are generated. They are potent detergents and may favor fusion [ 23 ]. In addition, arachidonic acid is the precursor of eicosanoids, prostanoids, leukotrienes and lipoxins, which may mediate further activation [ 24 ] and PLA2-induced hydrolysis of ether lipids gives rise to paf-acether [ 25 ]. It is possible that the interaction between gp120 and CD4 specifically modifies the cell membrane locally, preparing it for fusion. We hypothesize that the gp120-CD4-co-receptor complex activates PLA2 through protein kinase C (PKC) and plays a critical role in the fusion of the membrane phospholipids of the host cells and gp41 before viral entry. Indeed, the complex formed by CD4 and p56lck acts as the major receptor for HIV-1, HIV-2 and SIV, delivering intracellular activating signals. This complex binds to the viral envelope glycoprotein gp120. Following this binding, chemokine engagement appears to be required to generate the fusion active form of the envelope protein. This may involve the formation of a gp120-CD4-chemokine receptor complex, in which engagement of the chemokine receptor is dependent on a CD4-induced conformational change in env gp120 [ 26 - 28 ] as previously defined for the number of parameters contributing to fusion, i.e., fusion glycoproteins and the host-cell receptors [ 29 ]. However, further investigations are required to determine the real binding site of IM28. It is possible that IM28 acts on virus replication to inhibit existing syncytia, as previously reported [ 14 ]. Therefore, although the biochemical basis of this phenomenon remains to be discovered, IM28 prevents and inhibits the cell-cell fusion induced by HIV-1, giving it additional beneficial effects. Since differential ability to incorporate or maintain envelope on the virion might account for the differences in cell-to-cell versus cell-free infections in primary isolates, further studies with a more quantitative assay available for determining fusion inhibition as previously described [ 33 , 34 ] may also provide us with a greater understanding of the HIV-1 envelope structure and the HIV entry process.
Discussion
IM28 is a potent new derivative of DHEA that can stop the replication of HIV-1 by inhibiting its reverse transcriptase activity [ 14 ]. Here, we show that IM28 can also prevent and inhibit the fusion of infected cells (TF228.1.16 cells) to naïve cells including 293/CD4+ cells, which are stably transfected with human CD4 and highly susceptible to HIV-1 infection, and SupT1 cells [ 15 , 16 ]. The fusion of 293/CD4+ cells with TF228.1.16 cells was completely inhibited by a lower dose of IM28 than was the fusion of SupT1 cells with TF228.1.16 cells (data not shown). The fusion of TF228.1.16 cells to H4CD4+ (CD4 positive glial cell line) cells obtained by transfection of human neuroglioma cells [ 17 ] is also inhibited by IM28 (not shown). Therefore, IM28 and dexamethasone may inhibit cell-cell fusion and recombination-induced fusion mediated by the HIV env protein.Although the precise site at which IM28 acts to inhibit cell-cell fusion remains unknown, our results suggest that IM28 fights the HIV-1 virus at a new site. It is possible that this drug interacts with phospholipase A2 (PLA2), which plays an important role in the entry of HIV virus in the host cell [ 18 , 19 ]. Indeed, dexamethasone, a glucocorticoid, can inhibit the HIV-1, HIV-2 and SIVmac251 envelope glycoproteins and activate PLA2. PLA2 is activated when the envelope glycoprotein interacts with CD4. Due to its local membrane-destabilizing effect, PLA2 may play an important role in preparing the cell membrane for fusion with the viral particle. Activated PLA2 hydrolyzes membrane phospholipids in the sn-2 position, producing arachidonic acid and lysophospholipids [ 20 ]. These biochemical events also have downstream effects; the membrane is destabilized locally [ 21 , 22 ], and arachidonic acid and lysophospholipids are generated. They are potent detergents and may favor fusion [ 23 ]. In addition, arachidonic acid is the precursor of eicosanoids, prostanoids, leukotrienes and lipoxins, which may mediate further activation [ 24 ] and PLA2-induced hydrolysis of ether lipids gives rise to paf-acether [ 25 ]. It is possible that the interaction between gp120 and CD4 specifically modifies the cell membrane locally, preparing it for fusion. We hypothesize that the gp120-CD4-co-receptor complex activates PLA2 through protein kinase C (PKC) and plays a critical role in the fusion of the membrane phospholipids of the host cells and gp41 before viral entry. Indeed, the complex formed by CD4 and p56lck acts as the major receptor for HIV-1, HIV-2 and SIV, delivering intracellular activating signals. This complex binds to the viral envelope glycoprotein gp120. Following this binding, chemokine engagement appears to be required to generate the fusion active form of the envelope protein. This may involve the formation of a gp120-CD4-chemokine receptor complex, in which engagement of the chemokine receptor is dependent on a CD4-induced conformational change in env gp120 [ 26 - 28 ] as previously defined for the number of parameters contributing to fusion, i.e., fusion glycoproteins and the host-cell receptors [ 29 ]. However, further investigations are required to determine the real binding site of IM28. It is possible that IM28 acts on virus replication to inhibit existing syncytia, as previously reported [ 14 ]. Therefore, although the biochemical basis of this phenomenon remains to be discovered, IM28 prevents and inhibits the cell-cell fusion induced by HIV-1, giving it additional beneficial effects. Since differential ability to incorporate or maintain envelope on the virion might account for the differences in cell-to-cell versus cell-free infections in primary isolates, further studies with a more quantitative assay available for determining fusion inhibition as previously described [ 33 , 34 ] may also provide us with a greater understanding of the HIV-1 envelope structure and the HIV entry process.Conclusion In conclusion, our data show that IM28, a potent new analog of DHEA, is able to prevent and to inhibit cell-cell fusion, an important step at the beginning of HIV infection of naive cells, this drug seems to display the required properties for an anti-HIV drug.
Conclusion
In conclusion, our data show that IM28, a potent new analog of DHEA, is able to prevent and to inhibit cell-cell fusion, an important step at the beginning of HIV infection of naive cells, this drug seems to display the required properties for an anti-HIV drug.Methods Cell lines Three cell lines were used: TF228.1.16, which is a BJAB cell line that stably produces functionally active HIV-1 envelope protein (BH-10 clone of HIV-1 LAI) [ 30 ]. 293/CD4+ (human embryonic kidney 293 cells which over express human CD4), obtained through the AIDS Research and Reference Reagent Program; and SupT1 cells, purchased from the American Type Culture Collection (Rockville, MD, USA). Reagents DHEA, dextran-sulfate (DXSF), dexamethasone, suramin, heparin, the mannose-specific lectin concanavalin A (ConA) and Rowell Park Memorial Institute (RPMI)-1640 medium were purchased from Sigma-Aldrich (St Quentin-Fallavier, France). Cells were cultured in complete medium containing L-gltamine, penicillin, streptomycin and fetal calf serum. All these reagents were purchased from Invitrogen (Eragny, France). IM28 was produced from DHEA as specified in its data sheet (INPI 0990847; Fr2792201; Wo0106666; CRPH, Gabon). Fusion and syncytia assays Cultured 293/CD4+ cells in complete medium were harvested by trypsinization. These cells (5 × 10 4 ) were combined with an equal number of TF228.1.16 cells in a 24-well plate and incubated overnight at 37°C in a humidified incubator with 5% carbon dioxide as described by Moore et al. 1993 [ 11 ]. Adherent cells were fixed and stained with diff-quick (Sigma-Aldrich) and then observed under a Leitz microscope. To examine the effect of IM28 on HIV-1 envelope glycoprotein-mediated fusion, 293/CD4+ cells were mixed with TF228.1.16 cells in the presence of IM28. As a positive control for fusion inhibition, cells were incubated in parallel with dexamethasone, ConA, heparin, suramin and dextran sulfate 10 000 or 500 000, compounds known to interfere with mannose residues of envelope glycoprotein on HIV infectivity and HIV and measles virus-induced cell fusion [ 31 , 32 ]. The inhibitory activity of IM28 on fusion of 293/CD4+ cells with TF228.1.16 cells is expressed as a function of concentration and was compared with the inhibitory activity of the above mentioned compounds that interact with the HIV envelope protein. Fusion was examined by light microscopy after co-cultivation for 32 h. The percentage fusion is the ratio of cell surface involved in syncytia to the total cell surface. Syncytia were defined as giant cells, with diameters more than four times bigger than those of single cells. Percentage fusion was divided into three classes: 56–00% of the surface covered by syncytial = fusion; partial inhibition of fusion: < 10% of the surface is covered by syncytial = P; inhibition of syncytia formation = I. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett' test. All analyses were performed using the Graph-Pad Prism ®  computer program. Only  P  < 0.05 was considered significant.
Methods
Cell lines Three cell lines were used: TF228.1.16, which is a BJAB cell line that stably produces functionally active HIV-1 envelope protein (BH-10 clone of HIV-1 LAI) [ 30 ]. 293/CD4+ (human embryonic kidney 293 cells which over express human CD4), obtained through the AIDS Research and Reference Reagent Program; and SupT1 cells, purchased from the American Type Culture Collection (Rockville, MD, USA).
Cell lines
Three cell lines were used: TF228.1.16, which is a BJAB cell line that stably produces functionally active HIV-1 envelope protein (BH-10 clone of HIV-1 LAI) [ 30 ]. 293/CD4+ (human embryonic kidney 293 cells which over express human CD4), obtained through the AIDS Research and Reference Reagent Program; and SupT1 cells, purchased from the American Type Culture Collection (Rockville, MD, USA).Reagents DHEA, dextran-sulfate (DXSF), dexamethasone, suramin, heparin, the mannose-specific lectin concanavalin A (ConA) and Rowell Park Memorial Institute (RPMI)-1640 medium were purchased from Sigma-Aldrich (St Quentin-Fallavier, France). Cells were cultured in complete medium containing L-gltamine, penicillin, streptomycin and fetal calf serum. All these reagents were purchased from Invitrogen (Eragny, France). IM28 was produced from DHEA as specified in its data sheet (INPI 0990847; Fr2792201; Wo0106666; CRPH, Gabon).
Reagents
DHEA, dextran-sulfate (DXSF), dexamethasone, suramin, heparin, the mannose-specific lectin concanavalin A (ConA) and Rowell Park Memorial Institute (RPMI)-1640 medium were purchased from Sigma-Aldrich (St Quentin-Fallavier, France). Cells were cultured in complete medium containing L-gltamine, penicillin, streptomycin and fetal calf serum. All these reagents were purchased from Invitrogen (Eragny, France). IM28 was produced from DHEA as specified in its data sheet (INPI 0990847; Fr2792201; Wo0106666; CRPH, Gabon).Fusion and syncytia assays Cultured 293/CD4+ cells in complete medium were harvested by trypsinization. These cells (5 × 10 4 ) were combined with an equal number of TF228.1.16 cells in a 24-well plate and incubated overnight at 37°C in a humidified incubator with 5% carbon dioxide as described by Moore et al. 1993 [ 11 ]. Adherent cells were fixed and stained with diff-quick (Sigma-Aldrich) and then observed under a Leitz microscope. To examine the effect of IM28 on HIV-1 envelope glycoprotein-mediated fusion, 293/CD4+ cells were mixed with TF228.1.16 cells in the presence of IM28. As a positive control for fusion inhibition, cells were incubated in parallel with dexamethasone, ConA, heparin, suramin and dextran sulfate 10 000 or 500 000, compounds known to interfere with mannose residues of envelope glycoprotein on HIV infectivity and HIV and measles virus-induced cell fusion [ 31 , 32 ]. The inhibitory activity of IM28 on fusion of 293/CD4+ cells with TF228.1.16 cells is expressed as a function of concentration and was compared with the inhibitory activity of the above mentioned compounds that interact with the HIV envelope protein. Fusion was examined by light microscopy after co-cultivation for 32 h. The percentage fusion is the ratio of cell surface involved in syncytia to the total cell surface. Syncytia were defined as giant cells, with diameters more than four times bigger than those of single cells. Percentage fusion was divided into three classes: 56–00% of the surface covered by syncytial = fusion; partial inhibition of fusion: < 10% of the surface is covered by syncytial = P; inhibition of syncytia formation = I.
Fusion and syncytia assays
Cultured 293/CD4+ cells in complete medium were harvested by trypsinization. These cells (5 × 10 4 ) were combined with an equal number of TF228.1.16 cells in a 24-well plate and incubated overnight at 37°C in a humidified incubator with 5% carbon dioxide as described by Moore et al. 1993 [ 11 ]. Adherent cells were fixed and stained with diff-quick (Sigma-Aldrich) and then observed under a Leitz microscope.To examine the effect of IM28 on HIV-1 envelope glycoprotein-mediated fusion, 293/CD4+ cells were mixed with TF228.1.16 cells in the presence of IM28. As a positive control for fusion inhibition, cells were incubated in parallel with dexamethasone, ConA, heparin, suramin and dextran sulfate 10 000 or 500 000, compounds known to interfere with mannose residues of envelope glycoprotein on HIV infectivity and HIV and measles virus-induced cell fusion [ 31 , 32 ]. The inhibitory activity of IM28 on fusion of 293/CD4+ cells with TF228.1.16 cells is expressed as a function of concentration and was compared with the inhibitory activity of the above mentioned compounds that interact with the HIV envelope protein. Fusion was examined by light microscopy after co-cultivation for 32 h. The percentage fusion is the ratio of cell surface involved in syncytia to the total cell surface. Syncytia were defined as giant cells, with diameters more than four times bigger than those of single cells. Percentage fusion was divided into three classes: 56–00% of the surface covered by syncytial = fusion; partial inhibition of fusion: < 10% of the surface is covered by syncytial = P; inhibition of syncytia formation = I.Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett' test. All analyses were performed using the Graph-Pad Prism ®  computer program. Only  P  < 0.05 was considered significant.
Statistical analysis
Data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett' test. All analyses were performed using the Graph-Pad Prism ®  computer program. Only  P  < 0.05 was considered significant.PAuthors' contributions D M coordinated and participated in the design of the study, statistical analysis and the drafting of the manuscript. V P-M carried out and participated in the biological tests. M-Y A carried out and participated in the biological tests. B O carried out and participated in the biological tests. E M participated in the design of the study, carried out the biological tests and participated in the drafting of the manuscript.
Authors' contributions
D M coordinated and participated in the design of the study, statistical analysis and the drafting of the manuscript. V P-M carried out and participated in the biological tests. M-Y A carried out and participated in the biological tests. B O carried out and participated in the biological tests. E M participated in the design of the study, carried out the biological tests and participated in the drafting of the manuscript.


Analysis of growth and division often involves measurements made on cell populations, which tend to average data. The value of single cell analysis needs to be appreciated, and models based on findings from single cells should be taken into greater consideration in our understanding of the way in which cell size and division are co-ordinated. Examples are given of some single cell analyses in mammalian cells, yeast and other microorganisms. There is also a short discussion on how far the results are in accord with simple models.

Introduction What is the point of single cell studies of the cell cycle? The simple answer is that they provide extra information that is not available from studies of cell populations. Without them a cell biologist can be misled. It is easiest for me to start with the theme of the extensive results on single cells of the fission yeast  Schizosaccharomyces pombe  with which I have worked since the mid-1950s. It was then a fairly obscure organism for physiological studies though it had a good genetic background found by U. Leupold in Bern [ 1 ]. Since then it has flourished and quite large international meetings are now devoted entirely to it. For those unfamiliar with it, it is like a scaled-up bacterial rod with division at a medial septum, unlike budding yeasts. One the early results on its growth came from a single cell study by Bayne-Jones and Adolph [ 2 ]. Here I need to make a small digression about references. They will be given in this article but there are much longer accounts of nearly all the topics in my recent 100-page review [ 3 ]. When I took up fission yeast in the mid-fifties, I used a new microscopic technique, which gave by optical interferometry the total dry mass of single growing cells as well as their volume [ 4 ]. Volume increased, approximately in an exponential curve, through the first three quarters of the cycle but then stayed constant for the last quarter between mitosis and division. But total dry mass increased approximately linearly through the whole cycle. This was the first demonstration of linear growth, and I was surprised.
Introduction
What is the point of single cell studies of the cell cycle? The simple answer is that they provide extra information that is not available from studies of cell populations. Without them a cell biologist can be misled.It is easiest for me to start with the theme of the extensive results on single cells of the fission yeast  Schizosaccharomyces pombe  with which I have worked since the mid-1950s. It was then a fairly obscure organism for physiological studies though it had a good genetic background found by U. Leupold in Bern [ 1 ]. Since then it has flourished and quite large international meetings are now devoted entirely to it. For those unfamiliar with it, it is like a scaled-up bacterial rod with division at a medial septum, unlike budding yeasts.Schizosaccharomyces pombeOne the early results on its growth came from a single cell study by Bayne-Jones and Adolph [ 2 ]. Here I need to make a small digression about references. They will be given in this article but there are much longer accounts of nearly all the topics in my recent 100-page review [ 3 ]. When I took up fission yeast in the mid-fifties, I used a new microscopic technique, which gave by optical interferometry the total dry mass of single growing cells as well as their volume [ 4 ]. Volume increased, approximately in an exponential curve, through the first three quarters of the cycle but then stayed constant for the last quarter between mitosis and division. But total dry mass increased approximately linearly through the whole cycle. This was the first demonstration of linear growth, and I was surprised.Early synchrony techniques by induction This period of the fifties was when attention in this field was largely focused on the successful synchronisation of  Tetrahymena  and  Chlorella  by periodic changes in their environment. Good synchronous cultures would mean that powerful biochemical techniques, often enzyme activity assays at that time, could be applied in a cell cycle context. In the next 15 years, induction synchrony was somewhat improved but the cell cycles were always and inevitably distorted. Methods were also developed to select out a fraction of an asynchronous culture in one stage of the cycle and grow it up separately (for example," membrane elution", where cells growing on a membrane come away at division). They produce less distortion but a much lower yield than induction. Because of what can be measured in synchronous cultures, they are the natural choice for the molecular biologist. But it is as well to remember their limitations. The distortions after induction have been mentioned, but even with selection synchrony there are problems. The main one is that they are, in practice, not all that synchronous. The selected cells come from more than a very narrow region of the cycle. Some of the variation can be reduced by a correction for asynchrony [ 5 ] but there is still cell-to-cell variation in cycle stage and this can obscure the fine detail of the cycle. Single cell measurements may help here.
Early synchrony techniques by induction
This period of the fifties was when attention in this field was largely focused on the successful synchronisation of  Tetrahymena  and  Chlorella  by periodic changes in their environment. Good synchronous cultures would mean that powerful biochemical techniques, often enzyme activity assays at that time, could be applied in a cell cycle context. In the next 15 years, induction synchrony was somewhat improved but the cell cycles were always and inevitably distorted. Methods were also developed to select out a fraction of an asynchronous culture in one stage of the cycle and grow it up separately (for example," membrane elution", where cells growing on a membrane come away at division). They produce less distortion but a much lower yield than induction.TetrahymenaChlorellaBecause of what can be measured in synchronous cultures, they are the natural choice for the molecular biologist. But it is as well to remember their limitations. The distortions after induction have been mentioned, but even with selection synchrony there are problems. The main one is that they are, in practice, not all that synchronous. The selected cells come from more than a very narrow region of the cycle. Some of the variation can be reduced by a correction for asynchrony [ 5 ] but there is still cell-to-cell variation in cycle stage and this can obscure the fine detail of the cycle. Single cell measurements may help here.Single cell analysis in yeast Returning to single cell analyses of fission yeast, volume growth was followed in finer detail by Mitchison and Nurse [ 6 ]. One part of this analysis, on films taken previously by Fantes [ 7 ], showed that increase in volume was not a simple exponential during the growth phase in the first three quarters of the cycle but rather two linear segments with a rate change point (RCP) between them. The position of the RCP showed a large cell-to-cell variation. An important moral here is that these two linear segments vanished into an apparent exponential increase in a "well synchronised" culture made by selection. Such a culture scarcely showed the plateau in growth during the last quarter of the cycle. This distinction between single cells and synchronous cultures does of course depend on the frequency and accuracy of the data points. If the points have too much scatter, the fine detail of the single cell linear patterns is lost. There is also a second RCP at the end of the cycle. A much more detailed analysis of populations of single cells followed on films was made by Sveiczer  et al.  [ 8 ] on fission yeast. A plot of extension growth against birth size has a strong negative slope. So also does a plot of cycle time against birth size. This has important implications for the definitions of "size control", discussed in that paper.
Single cell analysis in yeast
Returning to single cell analyses of fission yeast, volume growth was followed in finer detail by Mitchison and Nurse [ 6 ]. One part of this analysis, on films taken previously by Fantes [ 7 ], showed that increase in volume was not a simple exponential during the growth phase in the first three quarters of the cycle but rather two linear segments with a rate change point (RCP) between them. The position of the RCP showed a large cell-to-cell variation. An important moral here is that these two linear segments vanished into an apparent exponential increase in a "well synchronised" culture made by selection. Such a culture scarcely showed the plateau in growth during the last quarter of the cycle. This distinction between single cells and synchronous cultures does of course depend on the frequency and accuracy of the data points. If the points have too much scatter, the fine detail of the single cell linear patterns is lost. There is also a second RCP at the end of the cycle.A much more detailed analysis of populations of single cells followed on films was made by Sveiczer  et al.  [ 8 ] on fission yeast. A plot of extension growth against birth size has a strong negative slope. So also does a plot of cycle time against birth size. This has important implications for the definitions of "size control", discussed in that paper.et al.Problems of single cell analysis Single cell studies have their problems. We have been lucky in using yeasts that are not apparently affected by growing on warm agar pads under a coverslip. They show "balanced growth", a property in which there is no change in extensive properties between successive cycles [ 9 ] and that should always be checked. Useful deductions can often be made with unbalanced growth but it will be a distortion of the normal cycle. The cells also have to keep still or be followed, a problem discussed below. We have not found ways of sticking yeast to glass (e.g. with lectins) that permit "normal" growth. Cells may also need a continuous supply of fresh medium, probably for oxygenation. Various types of microscopic mounting chambers have been described in the last 50 years or so, e.g. [ 10 ], but few seem to have been stringently tested. Many experimental studies on cell growth kinetics can be tedious; single cell studies are no exception. Here, however, modern automation is beginning to have very promising prospects. Anyone who has spent a day on a yeast film re-focusing the microscope every 5 min will welcome auto-focusing devices that are now available. Analysis has also become much easier with electronic imaging followed by image analysis programmes, and perhaps presentation on spreadsheets. It is now possible to have a programme that requires some hand work in the initial setting up under the microscope but will then run automatically, measuring cell length and diameter. This has been done for fifty or more single cells of fission yeast – a long way from the early days of using a ruler to measure the length of yeast cells on projected film images. Another point that should be raised here is that the new technology could profitably be applied to the growth of  Escherichia coli . The limitations of synchronous cultures in hiding the fine detail of increases in volume or area could well mean that single cell studies might reveal more than an exponential increase. There might even be something like the two linear patterns that were popular models in earlier work with this bacterium [ 11 ].
Problems of single cell analysis
Single cell studies have their problems. We have been lucky in using yeasts that are not apparently affected by growing on warm agar pads under a coverslip. They show "balanced growth", a property in which there is no change in extensive properties between successive cycles [ 9 ] and that should always be checked. Useful deductions can often be made with unbalanced growth but it will be a distortion of the normal cycle.The cells also have to keep still or be followed, a problem discussed below. We have not found ways of sticking yeast to glass (e.g. with lectins) that permit "normal" growth. Cells may also need a continuous supply of fresh medium, probably for oxygenation. Various types of microscopic mounting chambers have been described in the last 50 years or so, e.g. [ 10 ], but few seem to have been stringently tested.Many experimental studies on cell growth kinetics can be tedious; single cell studies are no exception. Here, however, modern automation is beginning to have very promising prospects. Anyone who has spent a day on a yeast film re-focusing the microscope every 5 min will welcome auto-focusing devices that are now available. Analysis has also become much easier with electronic imaging followed by image analysis programmes, and perhaps presentation on spreadsheets. It is now possible to have a programme that requires some hand work in the initial setting up under the microscope but will then run automatically, measuring cell length and diameter. This has been done for fifty or more single cells of fission yeast – a long way from the early days of using a ruler to measure the length of yeast cells on projected film images. Another point that should be raised here is that the new technology could profitably be applied to the growth of  Escherichia coli . The limitations of synchronous cultures in hiding the fine detail of increases in volume or area could well mean that single cell studies might reveal more than an exponential increase. There might even be something like the two linear patterns that were popular models in earlier work with this bacterium [ 11 ].Escherichia coliWhat to measure Volume and area of a rod-shaped organism are two of the parameters that can be measured in single growing cells. So is dry mass by interferometry. But there others, of which one of the most interesting is the use of the Cartesian diver, which was originally developed some fifty years ago at the Carlsberg Laboratory in Copenhagen. It requires technical skills and very tightly controlled temperature in water baths, but it is exquisitely sensitive. It can be used in at least two ways. One is as a diver balance, which measures "reduced weight" or weight in water. Providing there are not major changes in chemical composition, this is proportional to total dry mass. It was used on single cells of  Amoeba proteus  in an important classic paper by Prescott [ 12 ] mentioned below. It can also be used with minute divers as a respirometer. Hamburger [ 13 ] measured oxygen uptake in  Acanthamoeba a nd CO 2  production in fission yeast (Hamburger  et al.  [ 14 ]), in both cases over several cell cycles starting with single cells – a remarkable achievement. In both cases, the results were elegant linear patterns with an RCP at division. Another interesting single cell method was the colorimetric enzyme assay of single yeast cells in microdrops [ 15 ]. This might have been developed with promise, but was not followed up, partly perhaps because the results differed from similar assays in synchronous cultures. One of the advantages of single cell work with yeasts is that they stay still on an agar pad so they can be followed for a couple of cycles before overlapping spoils the image. This is not true of many mammalian cells, which move around on the substrate. One solution to this problem comes in the work on fibroblasts (mouse L cells) described in Zetterberg [ 16 ], Killander and Zetterberg [ 17 ], Zetterberg and Killander, [ 16 ]. These are part of an impressive body of work initiated using optical machinery gathered by Trigvar Caspersson, along with a great deal of skill and hard work. In one set of experiments on single cells [ 17 ], they made a measurement of the dry mass of single cells by interferometry and then placed it in the cycle by following it as it moved about until it divided. The difference in timing between the measurement and cell division gave the timing in the cycle. A second set of experiments used frequency analysis to set the cycle stages. This is a method widely used to determine G1, S and G2 in flow cytometry but is less suitable for the slow and imprecise doublings in something like dry mass. I therefore regard the single cell analyses as more reliable and they are not the same as those from the second method. What are needed now are techniques that combine the subtlety and precision of single cell measurements with the new techniques of automation. A promising start was made by Zicha and Dunn [ 19 ], and the development is being actively pursued elsewhere. Organisms which tend to be forgotten about these days are those lower eukaryotes that make poor material for molecular biologists because of inadequate genetic backgrounds. One important set of results are those from the early pioneer work of Prescott [ 12 ] on  Amoeba proteus  mentioned above. The results showed that the increase of single cell "dry mass" fell in a reverse exponential, with a rapid increase at the start of the cycle falling to zero towards the end. This, of course, is lethal for anyone who believes that a rising exponential is the paradigm for the cell cycle. Tetrahymena pyriformis  has a long and distinguished history in the cell cycle with its early induction synchrony. But in the 1960s there was a burst of studies on selected single cells or small groups. The growth patterns were often not well defined but it seems that absolute measurements of volume and of respiration rate were a better fit to linear growth (Prescott, [ 20 ]). Such analyses might now be checked using some of the semi-automated procedures referred to above.
What to measure
Volume and area of a rod-shaped organism are two of the parameters that can be measured in single growing cells. So is dry mass by interferometry. But there others, of which one of the most interesting is the use of the Cartesian diver, which was originally developed some fifty years ago at the Carlsberg Laboratory in Copenhagen. It requires technical skills and very tightly controlled temperature in water baths, but it is exquisitely sensitive. It can be used in at least two ways. One is as a diver balance, which measures "reduced weight" or weight in water. Providing there are not major changes in chemical composition, this is proportional to total dry mass. It was used on single cells of  Amoeba proteus  in an important classic paper by Prescott [ 12 ] mentioned below. It can also be used with minute divers as a respirometer. Hamburger [ 13 ] measured oxygen uptake in  Acanthamoeba a nd CO 2  production in fission yeast (Hamburger  et al.  [ 14 ]), in both cases over several cell cycles starting with single cells – a remarkable achievement. In both cases, the results were elegant linear patterns with an RCP at division.Amoeba proteusAcanthamoeba aet al.Another interesting single cell method was the colorimetric enzyme assay of single yeast cells in microdrops [ 15 ]. This might have been developed with promise, but was not followed up, partly perhaps because the results differed from similar assays in synchronous cultures.One of the advantages of single cell work with yeasts is that they stay still on an agar pad so they can be followed for a couple of cycles before overlapping spoils the image. This is not true of many mammalian cells, which move around on the substrate. One solution to this problem comes in the work on fibroblasts (mouse L cells) described in Zetterberg [ 16 ], Killander and Zetterberg [ 17 ], Zetterberg and Killander, [ 16 ]. These are part of an impressive body of work initiated using optical machinery gathered by Trigvar Caspersson, along with a great deal of skill and hard work. In one set of experiments on single cells [ 17 ], they made a measurement of the dry mass of single cells by interferometry and then placed it in the cycle by following it as it moved about until it divided. The difference in timing between the measurement and cell division gave the timing in the cycle. A second set of experiments used frequency analysis to set the cycle stages. This is a method widely used to determine G1, S and G2 in flow cytometry but is less suitable for the slow and imprecise doublings in something like dry mass. I therefore regard the single cell analyses as more reliable and they are not the same as those from the second method.What are needed now are techniques that combine the subtlety and precision of single cell measurements with the new techniques of automation. A promising start was made by Zicha and Dunn [ 19 ], and the development is being actively pursued elsewhere.Organisms which tend to be forgotten about these days are those lower eukaryotes that make poor material for molecular biologists because of inadequate genetic backgrounds. One important set of results are those from the early pioneer work of Prescott [ 12 ] on  Amoeba proteus  mentioned above. The results showed that the increase of single cell "dry mass" fell in a reverse exponential, with a rapid increase at the start of the cycle falling to zero towards the end. This, of course, is lethal for anyone who believes that a rising exponential is the paradigm for the cell cycle.Amoeba proteusTetrahymena pyriformis  has a long and distinguished history in the cell cycle with its early induction synchrony. But in the 1960s there was a burst of studies on selected single cells or small groups. The growth patterns were often not well defined but it seems that absolute measurements of volume and of respiration rate were a better fit to linear growth (Prescott, [ 20 ]). Such analyses might now be checked using some of the semi-automated procedures referred to above.Tetrahymena pyriformisGrowth in syncytia Physarum polycephalum  is a myxomycete of considerable importance in some earlier work on cell cycle control. It is effectively a big single multinucleate cell with complete natural synchrony in nuclear division. It does not show exponential increase in macromolecular synthesis. For instance, there are two peaks in the rate of protein synthesis, one in the S period and the other in G2 (Mittermayer  et al , [ 21 ]).
Growth in syncytia
Physarum polycephalum  is a myxomycete of considerable importance in some earlier work on cell cycle control. It is effectively a big single multinucleate cell with complete natural synchrony in nuclear division. It does not show exponential increase in macromolecular synthesis. For instance, there are two peaks in the rate of protein synthesis, one in the S period and the other in G2 (Mittermayer  et al , [ 21 ]).Physarum polycephalumet alGeneral conclusion It would appear that there are no universal patterns of growth in these lower eukaryotes.
General conclusion
It would appear that there are no universal patterns of growth in these lower eukaryotes.Models My title makes mention of "some models". Let me be clear that there are two quite different types of cell cycle models. One type includes detailed mathematical and molecular models dealing with discrete periodic events like mitosis (e.g. [ 22 ]). These are complex and can illustrate the relations between many components of a network at the event, on reasonable assumptions. They are important aids in understanding the events and are a fairly recent development in the cell cycle world. There are certain limitations at present. With mitosis, the models have problems with the starting event (a size control?), with location in cellular compartments, and with the final mechanical events. However, such models will certainly develop. However, what I am concerned with here are much earlier and much simpler models, not of periodic events in the cycle like DNA synthesis, but of continuous growth. Here the two dominant models were, for simplicity, an exponential pattern of increase and a linear one. My own view [ 3 ] of the earlier experiments is that, on the whole, they favour linear increase but it was also clear that some patterns, e.g. volume in fission yeast, are more complex. Linear increases with rate change points have certainly survived in fission yeast where there are no exponential increases (Table 1 in [ 3 ]) and this has revived for me an old hypothesis of "gene dosage". What, for instance, happens to synthesis rates between G1 and G2? But one thing is clear – that a single unifying dream of exponential synthesis is not in accord with the facts. It is really useless to wave Occam's Razor around. The end of his razor blade is "without necessity". In all reasonable judgements, the necessity is there. Beyond that is prejudice. Figure 1 Modes of growth in cell length of wild-type and wee1 mutant cells of fhe fission yeast  Schizosaccharomyces pombe , after Sweiczer et al. (1996) Figure 2 Growth through one cycle of individual Amoeba, after Prescott (1976)
Models
My title makes mention of "some models". Let me be clear that there are two quite different types of cell cycle models. One type includes detailed mathematical and molecular models dealing with discrete periodic events like mitosis (e.g. [ 22 ]). These are complex and can illustrate the relations between many components of a network at the event, on reasonable assumptions. They are important aids in understanding the events and are a fairly recent development in the cell cycle world. There are certain limitations at present. With mitosis, the models have problems with the starting event (a size control?), with location in cellular compartments, and with the final mechanical events. However, such models will certainly develop.However, what I am concerned with here are much earlier and much simpler models, not of periodic events in the cycle like DNA synthesis, but of continuous growth. Here the two dominant models were, for simplicity, an exponential pattern of increase and a linear one. My own view [ 3 ] of the earlier experiments is that, on the whole, they favour linear increase but it was also clear that some patterns, e.g. volume in fission yeast, are more complex. Linear increases with rate change points have certainly survived in fission yeast where there are no exponential increases (Table 1 in [ 3 ]) and this has revived for me an old hypothesis of "gene dosage". What, for instance, happens to synthesis rates between G1 and G2? But one thing is clear – that a single unifying dream of exponential synthesis is not in accord with the facts. It is really useless to wave Occam's Razor around. The end of his razor blade is "without necessity". In all reasonable judgements, the necessity is there. Beyond that is prejudice.