Cytokines involved in monocyte mediated tumor cell death and growth inhibition in serum-free medium.

In a serum-free culture system, the release of TNF, lI-1, lI-6, IFN-alpha, and IFN-beta during interaction of elutriated human monocytes (MO) with human tumor cells (TC) was studied by ELISA-technique. Contributions of these cytokines to inhibition of TC-growth and to induction of TC-death by supernatants (SU) gained from such MO/TC-interaction cultures were investigated using affinity chromatography for removal of individual cytokines. Although the TC used are relatively insensitive to recombinant human TNF, withdrawal of TNF causes 50% to 75% reduction of SU-induced TC-death rates, suggesting that susceptibility to TNF is raised during MO/TC-interaction by the other cytokines. Individual removal of other cytokines does not cause reduction of SU-mediated TC-death. However, combined withdrawal of lI-1 and IFN-alpha/beta causes in 2 of 4 TC-lines significant reduction of TC-death. Combined removal of TNF, IFN-alpha/beta, lI-1, and lI-6 leads to complete prevention of SU-mediated growth inhibitory and lytic effects, suggesting that besides these cytokines other signals are not involved significantly. SU-effects can be mimicked by appropriate combinations of authentic cytokines. The response of TC to SU- or cytokine-exposure is strikingly dependent on TC-density, leading at subconfluent TC-density exclusively to inhibition of growth and at postconfluent TC-density to induction of cell death. The principal effect of SU or cytokine combinations in this context seems to be the activation of growth inhibitory signal transduction pathways leading to TC-death in postconfluent TC-populations exclusively if growth stimulatory pathways are activated at the same time. Mouse L cells do not follow this reaction pattern: Their death is exclusively dependent on the presence of TNF in SU and they die upon SU-exposure at postconfluent as well as at subconfluent cell density.     

Suppression of tumor cell susceptibility to monocyte-induced cell death by growth-inhibitory signals generated during monocyte/tumor cell interaction

In a recently established serum-free in vitro system it has been demonstrated that the susceptibility of various human tumor cells to the induction of cell death by elutriated human monocytes is critically dependent on tumor cell density and growth state. In the present work it is shown by flow cytofluorometric analysis of bromodeoxyuridine incorporation rates and of expression of the proliferation-associated nuclear antigen Ki-67, that tumor cells forced out of the cell cycle into the quiescent state (G0), which can be accomplished by treatment with supernatant from monocyte/tumor cell interaction cultures, are no longer susceptible to the induction of cell death by monocytes. This suggests that processes essential for the lytic pathway cannot take place in quiescent cells. It is furthermore demonstrated that tumor cells are driven into G0 during interaction with monocytes and that the rate of transit from G1 to G0 increases with increasing monocyte dosage. This explains our earlier finding that maximum rates of tumor cell death are induced at rather low monocyte:tumor cell ratios of around 1:2 and that lysis is suppressed at higher monocyte dosages (van der Bosch et al.:Exp Cell Res 187:185-192, 1990). The potential significance of these findings for the supposed function of mononuclear phagocytes in tumor defense lies in the notion that tumor cells driven into G0 might escape this control and that signals involved in monocyte/tumor cell-interaction contribute to the accumulation of tumor cells in G0.     

Human T cell clones exerting multiple cytolytic activities show heterogeneity in susceptibility to inhibition by monoclonal antibodies

Human cytotoxic T cell clones (CTL) were obtained by limiting dilution after in vitro priming against an allogeneic Epstein Barr virus (EBV)-transformed B cell line (B-LCL) BSM. Three OKT3+, OKT8+ E rosette-forming (RFC) but EA gamma-RFC- clones with cytotoxic activity against the stimulator cell and one "non-cytolytic" clone were expanded for over 50 generations and further characterized. Clone G9 showed allospecific lysis of Cw3+ lymphocytes and B cell lines. Three cytolytic clones (G9, D11, and A3) showed cytotoxicity to the stimulator B-LCL, to the human plasma cell leukemia-derived line LICR-LON-HMY2 and to short-term cultured melanoma cells (O-mel). Four other EBV-transformed B-LCL unrelated to the stimulator B-LCL were not lysed. These clones also exerted cytotoxic activity against NK-sensitive target cells (TC), e.g., the erythroleukemia cell line K562. Other NK-sensitive TC, e.g., lymphoma-derived Daudi cells, were killed provided they were pretreated with phytohemagglutinin (PHA). Cytolytic activity against the B-LCL cell LICR-LON and O-mel, but not against K562 or PHA-treated target cells, was inhibited by monoclonal anti-HLA ABC antibodies (MCA). The cytolytic activities of OKT3+,8+ clones G9 and A3 but not that of OKT3+,8+ clone D11 were inhibited by OKT8. Another MCA, 13.3, directed against the murine glycoprotein T-200, inhibited the cytolytic activity of clone D11 against K562 but not against the stimulator cells. Clone G9 was not inhibited by MCA 13.3. The four clones, including the OKT4+ "non-cytotoxic" clone K12, exerted lytic activity against TC that are normally resistant to lysis provided these TC were pretreated with PHA. The TC specificity range of the clones was confirmed by cold target inhibition experiments. A correlation between blocking of lytic activity by cold TC and the percentage of conjugate formation with the particular cold TC was observed. Because these clones also show differential susceptibility to inhibition of lysis by various MCA, it is concluded that human cytotoxic T cell clones can exert multiple lytic activities, i.e., the operationally defined lytic mechanisms differ at least at certain stages of the lytic cycle.     

Target cell susceptibility to immune lysis and expression of MHC antigens are independent of position in the cell cycle

We have investigated the effects of progression through the mitotic cell cycle upon the tumor (P815) cell line's expression of H-2 antigens and susceptibility to immune cytolysis. By using a combination of DNA quantitation by Hoechst 33342 staining and fluorescence-activated cell sorter analysis we were able to obtain pure populations of G1, S, and G2+M cells for study. Furthermore, by employing a fluorescence quenching technique utilizing bromodeoxyuridine substitution we were able to monitor the times required for the P815 cells to transit each of the various phases. By using mAb and cloned CTL populations with well-defined specificities we observed that neither H-2 antigen expression nor lytic susceptibility to mAb plus complement or CTL fluctuated with cell cycle progression. Each of the three major MHC loci (H-2Kd, -Dd, and -Ld) was analyzed. These results demonstrated that expression of H-2 antigens and susceptibility to immune lysis were independent of a cell's position in the cell cycle.     

Growth inhibitory polypeptides in the regulation of cell proliferation

The growth of cells in culture and in vivo is modulated by different effectors, some of which are called growth factors. This designation is given to polypeptides that have the ability to enhance cellular growth. Other important growth regulatory molecules are the growth inhibitory polypeptides. The balance between stimulatory and inhibitory signals is evidently essential for normal control of cell proliferation. Disturbances of cellular growth thus presumably result from quantitative alterations between stimulatory and inhibitory signals that the cells get from their environment via their cell surface receptors. Thus, either enhanced amounts of stimulatory or decreased inhibitory signals can contribute to augmented, cancerous growth. An important growth regulator appears to be transforming growth factor-beta (TGF beta), which has both stimulatory and inhibitory effects on cells. The significance of growth inhibitors in the regulation of cellular growth and differentiation is becoming an important research field of modern biology.     

CTLs directed against HER2 specifically cross-react with HER3 and HER4

The human epidermal growth factor receptor 2 (HER2) has been targeted as a breast cancer-associated Ag by T cell-based immunotherapeutical strategies such as cancer vaccines and adoptive T cell transfer. The prerequisite for a successful T cell-based therapy is the induction of T cells capable of recognizing the HER2-expressing tumor cells. In this study, we generated human cytotoxic T cell clones directed against the HER2(369-377) epitope known to be naturally presented with HLA-A*0201. Those HER2-reactive CTLs, which were also tumor lytic, exhibited a similar lysis pattern dividing the targets in lysable and nonlysable tumor cells. Several HER2-expressing tumor cells became susceptible to CTL-mediated lysis after IFN-gamma treatment and, in parallel, up-regulated molecules of the Ag-presenting machinery, indicating that the tumor itself also contributes to the success of CTL-mediated killing. Some of the HER2(369-377)-reactive T cells specifically cross-reacted with the corresponding peptides derived from the family members HER3 and/or HER4 due to a high sequence homology. The epitopes HER3(356-364) and HER4(361-369) were endogenously processed and contributed to the susceptibility of cell lysis by HER cross-reacting CTLs. The principle of "double" or "triple targeting" the HER Ags by cross-reacting T cells will impact the further development of T cell-based therapies.     

Activation or tolerance of natural killer cells is modulated by ligand quality in a nonmonotonic manner

Natural killer (NK) cells extend important immune resistance in vertebrates by lysing infected and tumor cells. A fine balance between opposing signals generated by a diverse set of stimulatory and inhibitory NK-cell receptors determines the fate of target cells interacting with the NK cells. We have developed a mathematical model involving membrane proximal initial signaling events that provides novel mechanistic insights into how activation of NK cells is modulated by the half-life of receptor-ligand interaction and ligand concentrations. We show that strong stimulatory ligands produce digital activation, whereas weaker stimulatory ligands can mediate inhibition by strengthening the signals generated by inhibitory ligands, as indicated in experiments in knockout mice. We find under certain conditions, counterintuitively, inhibitory receptors can help mediate activation instead of inhibition. Mechanistic insights gained from NK-cell signaling can facilitate understanding of complex signaling responses that occur due to cross talk between dueling signaling pathways in other cell types.     

Heparin-binding growth factor/prostatropin attenuates inhibition of rat prostate tumor epithelial cell growth by transforming growth factor type beta

Summary Normal rat prostate epithelial cell growth requires both epidermal growth factor and heparin-binding growth factor/prostatropin. In contrast, epithelial cells derived from the transplantable Dunning R3327H rat tumor require either epidermal growth factor or heparin-binding growth factor/prostatropin. Transforming growth factor type beta inhibited normal epithelial cell growth. Transforming growth factor beta inhibited epidermal growth factor-dependent growth of tumor epithelial cells, independent of epidermal growth factor concentrations. Transforming growth factor beta increased the effective dose of heparin-binding growth factor type 1 required to support tumor epithelial cell growth by 10-fold but saturating levels of heparin-binding growth factor type 1 (290 pM) completely attenuated the inhibitory effect of transforming growth factor beta. These results suggest that prostate tumor epithelial cells may escape the inhibitory effect of transforming growth factor beta as a consequence of alteration of the concurrent requirement for both epidermal growth factor (or homologues) and heparin-binding growth factors. This work was supported by NCI Grant CA37589. Editor’s Statement The observation that heparin-binding growth factor/prostatropin can counteract the inhibitory effect of transforming growth factor beta in prostate epithelial cells may help explain how some cancers avoid the action of growth inhibitors and provides a model for studying how inhibitory peptides overcome the stimulatory signals generated by growth factors.     

Bacteriolytic effect of cessation of glucosamine supply, induced by specific inhibition of glucosamine-6-phosphate synthetase

The antibiotic tetaine (bacilysin) and its C-terminal epoxyaminoacid--anticapsin--are powerful inhibitors of glucosamine-6-phosphate synthetase (EC 5.3.1.19.) in cell-free extracts of Escherichia coli K-12. Tetaine acts on growing cells as a bactericidal agent. This bactericidal action, measured from 10 to 160 muM concentration, is a consequence of the induction of lysis of growing cells. The induction of lysis by tetaine is compared with the lytic action of some beta-lactams. Hypertonic medium, destruction of the antibiotic, presence of chloramphenicol or the addition of N-acetylglucosamine protect E. coli K-12 cells against lysis induced by tetaine. These effects are compared with those observed in the presence of penicillin G. The results indicate that inhibition of early or late stages of peptidoglycan synthesis all result in more or less the same consequence, i.e. death via cell lysis.     

From growth arrest to growth suppression.

Since the introduction of the cell cycle concept two approaches to study growth regulation of cells have been proposed. One claims that cells are naturally quiescent, requiring a stimulatory encouter with growth factors for induction of cell division. The other considers cellular multiplication as the natural steady-state; cessation of multiplication is thus a restriction imposed on the system. In the latter case emphasis is mainly on the signals involved in arrest of multiplication. This Prospect focuses on specific events occurring in mammalian cells at growth arrest, senescence, and terminal differentiation, specifically emphasizing the growth inhibitory factors, tumor suppressor genes, and other signals for growth suppression.     

Density-dependent tumor cell death and reversible cell cycle arrest: mutually exclusive modes of monocyte-mediated growth control

The population development of five human tumor cell lines is examined under the influence of elutriator-prepared human monocytes in a serum-free hormone- and growth factor-supplemented medium. Analysis was performed by electronic counting and sizing of tumor cell nuclei and flow cytometric detection of cell cycle phases. Tumor cell death is triggered at rather low monocyte:tumor cell ratios (1:2 to 1:4) whereas it is strongly reduced at high monocyte densities. Furthermore, it is shown that confluence of the target cell population is a necessary prerequisite for lysis. The data suggest that in monocyte/tumor cell cocultures the decision on target cell lysis is not made by the effector cell, but rather by the target cell and that the criterion for this decision is the target cell's ability or inability to respond to a monocyte challenge by arresting the cell cycle in G1. Interactions between target cells play an important role in determining the result of this decision process. A common basis is suggested for this kind of density-dependent monocyte-triggered lysis and density-dependent cell death in 3T3 cell cultures as described previously.     

Cytokines alter target cell susceptibility to lysis. II. Evaluation of tumor infiltrating lymphocytes

We studied the susceptibility of autologous and allogeneic tumors to lysis by human tumor infiltrating lymphocytes (TIL) after pre-incubation of the tumors with human rIFN-gamma and human rTNF-alpha. Preincubation of the tumor lines with IFN-gamma or TNF enhanced susceptibility to lysis significantly; the combination of both cytokines was more effective than either alone. Pretreatment for at least 24 h was required to enhance lytic susceptibility and maximal lysis was observed after pretreatment for 48 to 72 h. Highly specific TIL lysed only their autologous tumor targets and failed to lyse cytokine pretreated allogeneic tumor cells. In TIL populations with varying specificity, cytokine pretreatment of targets enhanced autologous lysis as well as allogeneic lysis. This cytokine-mediated effect could also be observed in a lectin-dependent cytotoxicity assay and did not correlate directly with enhanced expression of MHC class I Ag or the adhesion molecules LFA-3 and ICAM-1. These results suggest that enhancement of lysis may occur at a postbinding stage by making the target cell more sensitive to the cytotoxic factors delivered by the killer cell. The fact that lysis of cytokine treated targets by cells with LAK activity was not enhanced suggests that cells with lymphokine-activated killer activity and tumor-derived T cells kill tumor targets via different mechanisms.     

Lysis of autologous tumor cells by blood lymphocytes activated in autologous mixed lymphocyte tumor cell culture — No correlation with the postsurgical clinical course

Summary Mixed lymphocyte tumor cell cultures (MLTC) were initiated with cells collected at the time of surgery from 62 patients with the following diagnoses: 12 squamous cell carcinoma, 14 adenocarcinoma of the lung, 17 osteosarcomas, and 16 soft tissue sarcomas. The lytic effect generated against autologous tumor cells was tested on the 7th day. These patients had been part of previous evaluation, which revealed that the lysis of autologous tumor cells by freshly collected lymphocytes correlated with the postsurgical clinical course. Patients with long survival exhibited autotumor lysis without previous activation. Blood lymphocytes of about half of the primarily nonreactive patients were stimulated for lytic function in MLTC. However, this parameter showed no correlation with the clinical course.     

Treatment of human colon carcinoma cell lines with anti-neoplastic agents enhances their lytic sensitivity to antigen-specific CD8+ cytotoxic T lymphocytes

Certain anti-neoplastic agents at subtoxic doses may exert immunomodulatory effects, which alter the expression of specific tumor cell surface molecules. We reasoned that potential increases in tumor cell surface markers, such as those important for facilitating effector-target contact, as well as triggering cell death pathways, might then improve antigen (Ag)-specific T-cell-mediated tumor cytolysis. Here, in a human colon carcinoma cell model in vitro, we examined whether the anti-neoplastic agents 5-fluorouracil (5-FU), CPT-11 or cisplatin (CDDP) could upregulate the expression of specific tumor cell surface markers, which may then enhance productive lytic interactions between CD8+ CTL and Ag-bearing tumor cells. Based on our earlier studies, IFN-gamma treatment was included as a control for sensitization to CTL-mediated lysis. Pretreatment of the SW480 primary colon carcinoma cell line with IFN-gamma, 5-FU, CPT-11 or CDDP enhanced ICAM-1 and Fas expression, resulting in Ag-specific CTL-mediated lysis involving Fas-dependent and -independent mechanisms. In contrast, pretreatment of the SW620 metastatic isolate, derived from the same patient, with IFN-gamma, CPT-11 or CDDP, but not 5-FU, enhanced ICAM-1 expression, resulting in Ag-specific CTL-mediated lysis via Fas-independent mechanisms only. Flow cytometric-based assays were then developed to measure the effects of drug treatment on caspase signaling and apoptosis incurred by tumor targets after interaction with CTL. We found that the lytic enhancement caused by drug treatment of SW480 or SW620 targets was accompanied by an increase in caspase-3-like protease activity. A peptide-based caspase inhibitor abrogated CTL-mediated apoptosis, suggesting that "chemomodulation" involved regulation of the caspase pathway. These results revealed for the first time an important role for components of the caspase pathway, such as caspase-3-like proteases, in the sensitization of human colon carcinoma cells by anti-neoplastic agents to Ag-specific CTL. Thus, certain anti-neoplastic agents may display unique immunoregulatory properties that facilitate human colon carcinoma death by engaging the lytic capacity of Ag-specific CTL, which may have implications for chemoimmunotherapy strategies.     

Analysis of cytostatic/cytotoxic lymphokines: relationship of natural killer cytotoxic factor to recombinant lymphotoxin, recombinant tumor necrosis factor, and leukoregulin

Previous results that were obtained by using supernatants from the co-culture of human peripheral blood lymphocytes and the natural killer susceptible cell line K562 strongly inhibited the growth of various tumor cell lines. No correlation was observed between the susceptibility of the target cell lines to growth inhibition and to lysis by natural killer cells. Rather the spectrum of cytostatic activity and the characteristics of the soluble factor were similar to those of leukoregulin (LRG), a recently described lymphokine. Because of the recent availability of recombinant tumor necrosis factor (TNF) and lymphotoxin (LT), we compare the target selectivity and mechanism of action of these (TNF, LT, LRG) factors with natural killer cytotoxic factor (NKCF). The pattern of target cell susceptibility to growth inhibition or cytolysis by the factors were quite distinct from the pattern observed when cells were exposed to NKCF. Furthermore, antibodies to rLT or rTNF had no effect on LRG cytostasis or NKCF lysis, arguing against a requirement for or synergistic interaction with low levels of LT or TNF. Some of the targets susceptible to LRG were growth inhibited but were not lysed, thereby distinguishing it from NKCF. Furthermore, LRG cytostasis was not inhibited by mannose-6-PO4 or rabbit antibodies to granule cytolysin, both of which block natural killer cytotoxic factor. Therefore, LRG appears to be a cytostatic factor produced by large granular lymphocytes in response to K562 that is distinct from NKCF, TNF, and LT. In addition, NKCF, rLT, rTNF, and LRG, although having cytotoxic/cytostatic activity, are distinct functional factors and may represent a family of lytic factors.     

Penicillin-induced changes in the cell wall composition of Staphylococcus aureus before the onset of bacteriolysis

To analyze if chemical cell wall alterations contribute to penicillin-induced bacteriolysis, changes in the amount, stability, and chemical composition of staphylococcal cell walls were investigated. All analyses were performed before onset of bacteriolysis i.e. during the first 60 min following addition of different penicillin G doses. Only a slight reduction of the amount of cell wall material incorporated after penicillin addition at the optimal lytic concentration was observed as compared to control cells. However, the presence of higher penicillin G concentrations reduced the incorporation of wall material progressively without bacteriolysis. Losses of wall material during isolation of dodecylsulfate insoluble cell walls were monitored to assess the stability of the wall material following penicillin addition. Wall material grown at the lytic penicillin concentration was least stable but about 30% of the newly incorporated wall material withstood even the harsh conditions of mechanical breakage and dodecylsulfate treatment. Dodecylsulfate insoluble cell walls were used for chemical analyses. While peptidoglycan chain length was unaffected in the presence of penicillin, other wall parameters were considerably altered: peptide cross-linking was reduced in the wall material synthesized after addition of penicillin; reductions from approx. 85% in controls to about 60% were similar for lytic and also for very high penicillin concentrations leading to nonlytic death. O-acetylation was also reduced after treatment with penicillin; this effect paralleled the occurence of subsequent bacteriolysis at different drug concentrations. The results are not consistent with hypotheses explaining penicillin-induced lysis as a result of an overall weakened cell wall structure or an overall activation of autolytic wall enzymes but not conflicting with the model that ascribes penicillin-induced bacteriolysis as the result of a very restricted, local perforation of the peripheral cell wall (murosome-induced bacteriolysis).Abbreviations CL Cross-linking - DNFB 2,4-dinitro-1-fluorobenzole - MIC Minimal inhibitory concentration - OD Optical density at 578 nm - PEN Penicillin G     

Inhibition of transformed T cell growth in vitro by monoclonal antibodies directed against distinct activating molecules

Stimulatory of antigen-specific murine T cell hybridomas with the appropriate antigen has been shown to cause lymphokine secretion and inhibition of spontaneous cell growth. In this study, the effect of cellular activation on the growth of transformed T cells, of known or unknown antigen specificity, was explored with stimulatory monoclonal antibodies (mAb) that recognize nonclonally distributed T cell surface molecules. Anti-CD3 antibodies stimulated interleukin 2 (IL-2) secretion while they inhibited murine and human T cell tumor growth in vitro. Both responses required external cross-linking of the anti-CD3 antibodies. Activation via two molecules that are not physically associated with the T cell antigen receptor, Thy-1 and Ly-6, also inhibited transformed T cell growth while inducing IL-2 secretion. Notably, an anti-Thy-1 mAb that did not cause the transformed T cells to secrete lymphokines failed to affect their growth, and in fact blocked the growth inhibition induced by the stimulatory mAb. Regardless of which stimulating mAb was used, IL-2 production and cell growth were inversely proportional, manifesting similar antibody dose-response curves. Activation of a T cell hybridoma with stimulatory mAb resulted in rapid lysis, as evidenced by the release of 51Cr and lactate dehydrogenase. Cell cycle analysis demonstrated that cellular activation was accompanied by a cell cycle block between the G1 and S phases, and probably a slowing of the transit of cells already in S. These results demonstrate that the growth of a spectrum of neoplastic T cells, murine and human, can be inhibited by what are normally growth-promoting signals for non-transformed T cells.     

p53 potentiation of tumor cell susceptibility to CTL involves Fas and mitochondrial pathways

In this study, we have investigated the mechanisms used by wild-type p53 (wtp53) to potentiate tumor cell susceptibility to CTL-mediated cell death. We report that wtp53 restoration in a human lung carcinoma cell line Institut Gustave Roussy (IGR)-Heu, displaying a mutated p53, resulted in up-regulation of Fas/CD95 receptor expression associated with an increase of tumor cell sensitivity to the autologous CTL clone, Heu127. However, when IGR-Heu cells were transfected with Fas cDNA, no potentiation to Heu127-mediated lysis was observed, indicating that induction of CD95 is not sufficient to sensitize target cells to CTL killing. Importantly, our data indicate that the effect of wtp53 on the Fas-mediated pathway involves a degradation of short cellular FLICE inhibitory protein resulting in subsequent caspase 8 activation. Furthermore, we demonstrate that wtp53 restoration also resulted in CTL-induced Bid translocation into mitochondria and a subsequent mitochondrial membrane permeabilization leading to cytochrome c release. These results indicate that tumor cell killing by autologous CTL can be enhanced by targeting degranulation-independent mechanisms via restoration of wtp53, a key determinant of apoptotic machinery regulation.     

Lysis of intact yeast cells and isolated cell walls by an inducible enzyme system of Arthrobacter GJM-1.

Bacterium Arthrobacter GJM-1 known in the literature as a good producer of alpha-mannanase was found to accumulate in the culture fluid lytic activities against viable yeast cells during growth on isolated cell walls or beta-glucan fractions of yeast. The accumulation of the lytic activities showed an inducible character. The lytic system produced in the medium containing baker's yeast cell walls was capable of complete solubiliaztion of cell wals in vitro. The system lysed viable cells of a number of yeast species and induced their conversion to protoplasts in an osmotically stabilized medium. The lytic system showed different pH and temperature optima when viable cells or isolated cell walls were used as substrates. The pH optimum of the lysis of isolated cell walls was identical with pH optimum of beta-glucanase activities in the crude system. The results pointed out that in the lysis of intact cells, in addition to beta-glucanases, some other factor is involved. Substantial differences in the nature of the outer and the inner surface of cell walls of Saccharomuces cerevisiae were confirmed in this paper based on the different susceptibility to lysis of the cell walls in vivo and in vitro.     

Lytic agents, cell permeability, and monolayer penetrability

Cell lysis induced by lytic agents is the terminal phase of a series of events leading to membrane disorganization and breadkdown with the release of cellular macromolecules. Permeability changes following exposure to lytic systems may range from selective effects on ion fluxes to gross membrane damage and cell leakage. Lysis can be conceived as an interfacial phenomenon, and the action of surface-active agents on erythrocytes has provided a model in which to investigate relationships between hemolysis and chemical structure, ionic charge, surface tension lowering, and ability to penetrate monolayers of membrane lipid components. Evidence suggests that lysis follows the attainment of surface pressures exceeding a "critical collapse" level and could involve membrane cholesterol or phospholipid. Similarities of chemical composition of membranes from various cell types could account for lytic responses observed on interaction with surface-active agents. Cell membranes usually contain about 20–30 % lipid and 50–75 % protein. One or two major phospholipids are present in all cell membranes, but sterols are not detectable in bacterial membranes other than those of the Mycoplasma group. The rigid cell wall in bacteria has an important bearing on their response to treatment with lytic agents. Removal of the wall renders the protoplast membrane sensitive to rapid lysis with surfactants. Isolated membranes of erythrocytes and bacteria are rapidly dissociated by surface-active agents. Products of dissociation of bacterial membranes have uniform behavior in the ultracentrifuge (sedimentation coefficients 2–3S). Dissociation of membrane proteins from lipids and the isolation and characterization of these proteins will provide a basis for investigating the specificity of interaction of lytic agents with biomembranes.