WI-38 Human Diploid Fibroblasts Research Articles

Increased stability of the p16 mRNA with replicative senescence.

Expression of p16(INK4a) is elevated during ageing and replicative senescence. Here, we report the presence of an instability determinant within the 3'-untranslated region (UTR) of the p16 messenger RNA in WI-38 human diploid fibroblasts. The p16 3'UTR was found to be a specific target of AUF1, an RNA-binding protein implicated in promoting mRNA decay. Both AUF1 levels and AUF1-p16 mRNA associations were strikingly more abundant in early-passage than late-passage fibroblast cultures. Moreover, short interfering RNA-based reductions in AUF1 levels increased the stability of p16 3'UTR-containing transcripts, elevated the expression of p16 and accentuated the senescence phenotype. Together, our findings show that p16 mRNA turnover decreases during replicative senescence and that the instability-conferring region is located within the 3'UTR of p16, as well as identifying AUF1 as a critical mediator of these regulatory events.

Inhibition of phosphatidylinostol 3-kinase uncouples H 2O 2-induced senescent phenotype and cell cycle arrest in normal human diploid fibroblasts

Exposure of WI38 human diploid fibroblasts (HDFs) to hydrogen peroxide (H 2O 2) induced premature senescence. The senescent HDFs were permanently arrested and exhibited a senescent phenotype including enlarged and flattened cell morphology and increased senescence-associated β-galactosidase (SA-β-gal) activity. The induction of HDF senescence was associated with an activation of p53, increased expression of p21 Cip1/WAF1, and hypophosphorylation of retinoblastoma protein (Rb), while no changes in the expression of p16 Ink4a, p27 Kip1, and p14 Arf were observed. Exposure of WI38 cells to H 2O 2 also selectively activated phosphatidylinostol 3-kinase (PI3 kinase) and mitogen-activated protein kinase (MAPK) kinase (MEK), while no changes in p38 MAPK and Jun kinase (JNK) activities were observed. Selective inhibition of PI3 kinase activity with LY294002 abrogated H 2O 2-induced cell enlargement and flattened morphology and significantly attenuated the increase in SA-β-gal activity, but did not affect H 2O 2-induced cell cycle arrest. In contrast, selective inhibition of MEK and p38 MAPK with PD98059 and SB203580, respectively, produced no significant effect on H 2O 2-induced senescent phenotype and cell cycle arrest. These findings demonstrate that expression of the senescent phenotype can be uncoupled from cell cycle arrest in prematurely senescent cells induced by H 2O 2 and does not contribute to the maintenance of permanent cell cycle arrest.

Regulation of Cyclin D1/Cdk4 Complexes by Calcium/Calmodulin-dependent Protein Kinase I

The selective inhibitor of the multifunctional calcium/calmodulin-dependent kinases (CaMK), KN-93, arrests a variety of cell types in G(1). However, the biochemical nature of this G(1) arrest point and the physiological target of KN-93 in G(1) remain controversial. Here we show that in WI-38 human diploid fibroblasts KN-93 reversibly arrested cells in late G(1) prior to detectable cyclin-dependent kinase 4 (cdk4) activation. At the KN-93 arrest point, we found that cyclin D1/cdk4 complexes had assembled with p21/p27, accumulated in the nucleus, and become phosphorylated on Thr-172, yet were relatively inactive. Additional examination of cdk4 complexes by gel filtration analysis demonstrated that, in late G(1), cyclin D1-containing complexes migrated toward lower molecular weight (M(r)) fractions and this altered migration was accompanied by the appearance of two peaks of cdk4 activity, at 150-200 and 70 kDa, respectively. KN-93 prevented both the activation of cdk4, and this shift in cyclin D1 migration and overexpression of cyclin D1/cdk4 overcame the KN-93 arrest. To determine which multifunctional CaMK acts in G(1), we expressed kinase-deficient forms of CaMKI and CaMKII. Overexpression of kinase-deficient CaMKI, but not CaMKII, prevented cdk4 activation, mimicking the KN-93 arrest point. Therefore, we hypothesize that KN-93 prevents a very late, uncharacterized step in cyclin D/cdk4 activation that involves CaMKI and follows complex assembly, nuclear entry, and phosphorylation.

Proteasome inhibition increases HuR level, restores heat-inducible HSP72 expression and thermotolerance in WI-38 senescent human fibroblasts

At the end of their replicative potential in vitro, late passage WI-38 human diploid fibroblasts (HDF) have a low basal expression of heat shock protein 72 (HSP72) and an attenuated ability to induce it in response to heat shock. The transient exposure to the specific and reversible proteasome inhibitor MG132 during a mild heat shock induced late passage HDF to synthesize and accumulate high levels of HSP72. This HSP72 expression was long-lasting and appeared to result from both increased cytoplasmic levels and enhanced translation of HSP72 mRNA. The level of HuR, a stabilizing mRNA-binding protein, increased following the MG132 treatment. This result is consistent with the proposed role of HuR in assisting mRNA export to the cytoplasm and in antagonizing its degradation. Furthermore, the previous exposure of late passage HDF to a mild heat shock in the presence of MG132 protected these cells against the otherwise lethal effect of a subsequent severe heat shock. This acquisition of thermotolerance appeared to be correlated with the level of HSP72.

Retrovirally mediated overexpression of peroxiredoxin VI increases the survival of WI-38 human diploid fibroblasts exposed to cytotoxic doses of tert-butylhydroperoxide and UVB.

In this work, stable overexpression of peroxiredoxin VI was generated in WI-38 human diploid fibroblasts using a retrovirus-mediated transfection system. Estimation of cell survival showed that peroxiredoxin VI provides a significant protection against tert-butylhydroperoxide- or UVB-caused cytotoxicity. No protection was found against ethanol- or H(2)O(2)-caused cytotoxicity. These effects are correlated with the known functions of Prx VI.

Oxidative DNA damage as a marker of aging in WI-38 human fibroblasts

Cause–effect relationships between oxidative stress, DNA damage and aging were investigated in WI-38 human diploid fibroblasts at 21, 41 or 58 population doublings (PDs), corresponding to young, middle age or old fibroblasts, respectively. Oxidative DNA damage was evaluated by immunohistochemical detection of 8-hydroxy-2′deoxyguanosine (8-OHdG) adducts or by single cell microgel electrophoresis (COMET assay). Aging was evaluated by growth rate, senescence-associated-β-galactosidase (SA-β galactosidase) activity, cell cycle distribution, and expression of p21. Our results demonstrate that (i) oxidative DNA damage is proportional to the age of cells (ii) DNA damage in old/58 PDs cells reflects both an increased susceptibility to oxidative stress, induced by acute exposure to sub-lethal concentrations of hydrogen peroxide (H 2O 2), and a reduced efficiency of repair mechanisms. We also show that mild chronic oxidative stress, induced by prolonged exposure to 5 μM H 2O 2, accelerates aging in fibroblasts. In fact, this treatment increased 8-OHdG levels, SA-β-galactosidase activity, and G0/G1 cell cycle arrest in middle age/41 PDs, making them similar to H 2O 2-untreated old/58 PDs cells. Although other mechanisms may concur in mediating the effects of H 2O 2, these results lend support to the concept that oxidative stress may be a key determinant of aging. Measurements of oxidative DNA damage might therefore be exploited as reliable marker of cellular aging.

Overexpression of apolipoprotein J in human fibroblasts protects against cytotoxicity and premature senescence induced by ethanol and tert-butylhydroperoxide.

Human diploid fibroblasts (HDFs) exposed to subcytotoxic stresses under H2O2, tert-butylhydroperoxide (t-BHP), and ethanol (EtOH) undergo stress-induced premature senescence (SIPS) characterized by many biomarkers of HDFs replicative senescence. Among these biomarkers are a growth arrest, an increase in the senescence-associated beta-galactosidase activity, a senescent morphology, an overexpression of p21waf-1 and the subsequent inability to phosphorylate pRb, the presence of the common 4977-bp mitochondrial deletion, and an increase in the steady-state level of several senescence-associated genes such as apolipoprotein J (apo J). Apo J has been described as a survival gene against cytotoxic stress. In order to study whether apo J would be protective against cytotoxicity SIPS and replicative senescence in human fibroblasts, a full-length complementary deoxyribonucleic acid of apo J was transfected into WI-38 HDFs and SV40-transformed WI-38 HDFs. The overexpression of apo J resulted in an increased cell survival after t-BHP and EtOH stresses at cytotoxic concentrations. In addition, when WI-38 HDFs were exposed to 5 subcytotoxic stresses with EtOH or t-BHP, in conditions that were previously shown to induce SIPS, a lower induction of 2 biomarkers of SIPS was observed in HDFs overexpressing apo J. No effect of apo J overexpression was observed on the proliferative life span of HDFs, even if apo J overexpression triggered osteonectin (SPARC) overexpression, which was shown to decrease the mitogenic potential of platelet-derived growth factor but not of other common growth-inducing conditions. Apo J senescence-related overexpression is proposed to have antiapoptotic rather than antiproliferative effects.

Growth kinetics rather than stress accelerate telomere shortening in cultures of human diploid fibroblasts in oxidative stress-induced premature senescence

WI-38 human diploid fibroblasts underwent accelerated telomere shortening (490 bp/stress) and growth arrest after exposure to four subcytotoxic 100 μM tert-butylhydroperoxide (t-BHP) stresses, with a stress at every two population doublings (PD). After subcytotoxic 160 μM H 2O 2 stress or five repeated 30 μM t-BHP stresses along the same PD, respectively a 322±55 and 380±129 bp telomere shortening was observed only during the first PD after stress. The percentage of cells resuming proliferation after stress suggests this telomere shortening is due to the number of cell divisions accomplished to reach confluence during the first PD after stress.

Regulation of Cyclin-Dependent Kinase 2 Activity by Ceramide

Cyclin-dependent kinases have been implicated in the inactivation of retinoblastoma (Rb) protein and cell cycle progression. Recent studies have demonstrated that the lipid molecule ceramide is able to induce Rb hypophosphorylation leading to growth arrest and cellular senescence. In this study, we examined the underlying mechanisms of Rb hypophosphorylation and cell cycle progression utilizing the antiproliferative molecule ceramide. C6-Ceramide induced a G0/G1 arrest of the cell cycle in WI38 human diploid fibroblasts. Employing immunoprecipitation kinase assays, we found that ceramide specifically inhibited cyclin-dependent kinase CDK2, with a mild effect on CDC2 and significantly less effect on CDK4. The effect of ceramide was specific such that C6-dihydroceramide was not effective. Ceramide did not directly inhibit CDK2 in vitro but caused activation of p21, a major class of CDK-inhibitory proteins, and led to a greater association of p21 to CDK2. Using purified protein phosphatases, we showed that ceramide activated both protein phosphatase 1 and protein phosphatase 2A activities specific for CDK2 in vitro. Further, calyculin A and okadaic acid, both potent protein phosphatase inhibitors, together almost completely reversed the effects of ceramide on CDK2 inhibition. Taken together, these results demonstrate a dual mechanism by which ceramide inhibits the cell cycle. Ceramide causes an increase in p21 association with CDK2 and through activation of protein phosphatases selectively regulates CDK2. These events may lead to activation of Rb protein and subsequent cell cycle arrest.

Induction of replicative senescence biomarkers by sublethal oxidative stresses in normal human fibroblast

We tested the long-term effects of sublethal oxidative stresses on replicative senescence. WI-38 human diploid fibroblasts (HDFs) at early cumulative population doublings (CPDs) were exposed to five stresses with 30 μM tert-butylhydroperoxide (t-BHP). After at least 2 d of recovery, the cells developed biomarkers of replicative senescence: loss of replicative potential, increase in senescence-associated β-galactosidase activity, overexpression of p21 Waf-1/SDI-1/Cip1, and inability to hyperphosphorylate pRb. The level of mRNAs overexpressed in senescent WI-38 or IMR-90 HDFs increased after five stresses with 30 μM t-BHP or a single stress under 450 μM H 2O 2. These corresponding genes include fibronectin, osteonectin, α1(I)-procollagen, apolipoprotein J, SM22, SS9, and GTP-α binding protein. The common 4977 bp mitochondrial DNA deletion was detected in WI-38 HDFs at late CPDs and at early CPDs after t-BHP stresses. In conclusion, sublethal oxidative stresses lead HDFs to a state close to replicative senescence.

Ceramide induces expression of the senescence histochemical marker, β-galactosidase, in human fibroblasts

We recently showed that ceramide is elevated in senescence and that when administered to low-passage cells induces biochemical changes characteristic of senescence. The in situ histochemical marker β-galactosidase (β-Gal) has provided an important tool in the study of cellular senescence. We investigated the ability of ceramide to induce the expression of β-Gal and correlated this with cell proliferation. We find that d-e-C 6-ceramide, induces the expression of acidic β-Gal in fetal lung-derived Wi-38 human diploid fibroblasts. Our results show that this induction is: (1) time and concentration dependent; and (2) reversible upon ceramide removal. We also find that concomitant with the onset of β-Gal staining, DNA synthesis is blocked. These conditions are reversible. The induction of β-Gal expression is specific to C 6-ceramide. We discuss a potential role of β-Gal in the regulation of senescence. Although signal transduction of senescence is still not fully understood, this new evidence strengthens the hypothesis that ceramide plays a key role in signaling down stream biochemical changes in cellular senescence.

Increase of the cellular growth of old human diploid fibroblasts by radical scavenger: methanolic extract of broad beans.

The methanolic extract from broad beans (MEBB), which is comprised of phenolic compounds, has free-radical scavenging activity. The effects of MEBB on cytosolic antioxidant enzymes and cell proliferation were examined in cultures of old (78-84% life-span completed) WI-38 human diploid fibroblasts. Because catechin is polyphenol and has radical scavenging activity, it was used as the control in experiments. We observed that MEBB increased cellular growth when added to the cell culture. In MEBB at 40 and 120 micrograms/mL, the cell proliferation increased by 14 and 27%, respectively, as compared to the control. In catechin, cell proliferation increased as well. Regarding cytosolic glutathione peroxidase (GSH-Px) activity, treatment of old cells with MEBB at 40 and 120 micrograms/mL resulted in decreases as compared to the control. In contrast, catechin showed no similarities to the modification of GSH-Px activity. Cytosolic SOD activity was increased by treatment with 40 micrograms/mL MEBB, and the activity showed a gradual decrease with increased MEBB concentrations. A similar trend occurred in the cells treated with catechin (4-20 microM). These results suggest that cytosolic antioxidant enzyme activities in old cells may be modulated by MEBB treatment. We conclude that there may be a relation between the optimum MEBB concentration for the increase of cellular growth and the MEBB concentration required to exhibit a decrease in GSH-Px activity.

Cell-cycle-dependent changes in ceramide levels preceding retinoblastoma protein dephosphorylation in G2/M.

Ceramide functions as a growth-inhibitory lipid-signalling molecule and might have a role in mediating the effects of extracellular agents on cell growth, differentiation and senescence. Here we investigate the roles of ceramide in cell cycle progression. With the use of the model of serum withdrawal, we were able to synchronize Wi-38 human diploid fibroblasts at different stages of cell cycle. Serum stimulation resulted in G0 to G1/S progression as determined by flow cytometric analysis and [3H]thymidine incorporation. Analyses of endogenous ceramide levels demonstrated that ceramide levels remained relatively constant on serum stimulation, indicating that ceramide might not be critical during G1/S transition. Treating exponentially growing Wi-38 human diploid fibroblasts with nocodazole led to cell cycle arrest at the G2/M phase of the cell cycle; 2 h after the removal of nocodazole, retinoblastoma (Rb) protein became dephosphorylated and the cells exited from G2/M and moved to the G1 phase of the new cycle. When cells were released from G2/M block by nocodazole, and before Rb protein dephosphorylation, endogenous ceramide levels transiently increased up to 2-fold at 0.5 h after the removal of nocodazole. Fumonisin B1, an inhibitor of ceramide synthase, inhibited the elevation of ceramide levels. Desipramine and SR33557, both acid sphingomyelinase inhibitors, did not have an appreciable effect on the elevation of ceramide levels. Furthermore, fumonisin B1 inhibited Rb protein dephosphorylation induced by endogenous ceramide but not by exogenous ceramide. These results demonstrate for the first time changes in ceramide during cell cycle progression and suggest that ceramide synthesized de novo might function as an endogenous modulator of Rb protein and cell cycle progression.

The Major Calpain Isozymes Are Long-lived Proteins: DESIGN OF AN ANTISENSE STRATEGY FOR CALPAIN DEPLETION IN CULTURED CELLS

Calpains are intracellular Ca2+-dependent proteases that are thought to participate in Ca2+-associated signal transduction pathways. It has been proposed that calpains are activated by an autoproteolytic mechanism. If this is true one would expect a relatively short half-life for calpain protein in cells. To test this hypothesis, WI-38 human diploid fibroblasts were pulse-labeled with [35S]methionine, and calpain was immunoprecipitated at various times after chasing with nonradioactive methionine to determine residual radioactivity. The results demonstrated that the two major calpain isozymes, m-calpain and micro-calpain, had metabolic half-lives of approximately 5 days. Calpains were long-lived proteins in several human cell lines, A-431, HeLa, VA-13, C-33A, and TE2 cells. In addition, calpastatin, the calpain-specific inhibitor protein, also had a long metabolic half-life. These observations suggest that the model for calpain activation by autoproteolysis requires re-investigation. Based on a knowledge of calpain metabolic stability, a protocol was devised for chronic exposure of WI-38 cells and HeLa cells to a calpain small subunit antisense oligodeoxyribonucleotide. Depletion of calpain small subunit after 5 or more days of treatment led to inhibition of cell proliferation that could be reversed by removal of antisense oligodeoxyribonucleotide from the culture medium. Together with previous studies, these results indicate a requirement for calpains in mammalian cell proliferation.

724 Acetylcholinesterase inhibitors increase the amount of retrograde transport of hrp in the rat masseter motoneuron

Skin fibroblasts (LNSV) derived from a hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient patient with the Lesch-Nyhan syndrome, who has glucose-6-phosphate dehydrogenase (G6PD) type A, were transformed with SV40 and hybridized with WI38 human diploid fibroblasts derived from a female embryo which have normal HGPRT and G6PD type B activities. The hybrid clones selected in hypoxanthine, aminopterin and thymidine (HAT) medium, were essentially tetraploid and contained three X and one Y chromosomes. These hybrids contained HGPRT, types A and B and the AB heteropolymeric form of G6PD enzymes which were indicative that in these cells X linked genes of both parental cells were fully active. Hybrids back-selected in medium containing 8-azaguanine (8-AG) contained only two X chromosomes. They had no HGPRT activity and they contained only G6PD type A enzyme. It is concluded that the hybrid cells which grew in the presence of 8-AG retained the X chromosome of the LNSV parental cell and apparently the inactive X of the WI 38 cell.

Role of Ceramide in Cellular Senescence

Recently the sphingomyelin cycle, involving the hydrolysis of membrane sphingomyelin by an activated sphingomyelinase to generate ceramide, has emerged as a key pathway in cell differentiation and apoptosis in leukemic and other cell types. Here we investigate a role for this pathway in the senescence of WI-38 human diploid fibroblasts (HDF). We found that endogenous levels of ceramide increased considerably (4-fold) and specifically (compared with other lipids) as cells entered the senescent phase. Investigation of the mechanism of increased ceramide led to the discovery that neutral sphingomyelinase activity is elevated 8-10 fold in senescent cells. There were no changes in sphingomyelinase activity or ceramide levels as HDF entered quiescence following serum withdrawal or contact inhibition. Thus, the activation of the sphingomyelinase/ceramide pathway in HDF is due to senescence and supports the hypotheses that senescence represents a distinct program of cell development that can be differentiated from quiescence. Additional studies disclosed the ability of ceramide to induce a senescent phenotype. Thus, when exogenous ceramide (15 microM) was administered to young WI-38 HDF, it produced endogenous levels comparable to those observed in senescent cells (as determined by metabolic labeling studies). Ceramide concentrations of 10-15 microM inhibited the growth of young HDF and induced a senescent phenotype by its ability to inhibit DNA synthesis and mitogenesis. These concentrations of ceramide also induced retinoblastoma dephosphorylation and inhibited serum-induced AP-1 activation in young HDF, thus recapitulating basic biochemical and molecular changes of senescence. Sphingomyelinase and ceramide may thus be implicated as mediators of cellular senescence.

Identification of Proteins Differentially Expressed in Quiescent and Proliferatively Senescent Fibroblast Cultures

We have examined the differential expression of proteins in quiescent (nongrowing) early versus late population doubling level (PDL) WI-38 human diploid fibroblasts. Age-associated changes in gene expression at the level of both secreted and total cellular protein were identified using high-resolution, two-dimensional gel electrophoresis followed by N-terminal sequencing or Western blot analysis. A comparison of secreted proteins revealed the senescence-specific absence of all forms of EPC-1, a protein associated with early PDL cells in the G0 state. This comparison also revealed the absence of a processed (cleaved) form of αI (type III) procollagen in senescent cells, suggesting a distinctive remodeling of the extracellular matrix. A comparison of total cellular protein between early passage and proliferatively aged fibroblasts identified an altered form of the enzyme ubiquitin carboxyl-terminal hydrolase in senescent cells, a result that could explain the increase in ubiquitinated proteins in these cells. These findings further elucidate the phenotype of fibroblasts aged in vitro and support the concept that senescent cells are arrested in a state fundamentally different from that of G0 early PDL cells.

A growth factor-inducible gene encodes a novel nuclear protein with zinc finger structure.

Growth factors rapidly induce numerous genes that encode regulatory proteins, many of which have been identified by cDNA cloning. In this study, differential hybridization was used to screen a cDNA library constructed from human mammary carcinoma MDA-468 cells that were stimulated with transforming growth factor beta-1 (TGF-beta 1) in the presence of cycloheximide. One of the cDNA clones that was induced by TGF-beta 1 was found to have a nucleotide sequence that predicts a 9,450-Da protein with homology to regulatory DNA-binding proteins. This clone was designated metallopan-stimulin-1 (MPS-1) because it encodes a metalloprotein whose mRNA is expressed in a wide variety of actively proliferating cells and tumor tissues. MPS-1 protein contains one "zinc finger" domain of the C4 type, similar to those present in proteins of the steroid/thyroid hormone receptor superfamily and other DNA-binding proteins. The mRNA for MPS-1 was induced in MDA-468 cells by fetal calf serum, TGF-beta 1, TGF-beta 2, and cAMP analogues. The MPS-1 gene is expressed at relatively high levels in several human carcinoma cell lines, particularly those derived from ectodermal layers, and at higher levels in melanomas (ontogenically of neural origin). In contrast, the MPS-1 mRNA is expressed at low levels in normal WI-38 human lung diploid fibroblasts in culture. We hypothesize that MPS-1 protein may play a role as a potentially important mediator of cellular proliferative responses to various growth factors and other environmental signals.

Effect of the Tumor Promoter Phorbol 12-Myristate 13-Acetate (PMA) on Proliferation of Young and Senescent WI-38 Human Diploid Fibroblasts

The effects of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on the proliferation, protein kinase C activity (PKC), and c- fos gene expression were examined in cultures of young and senescent (90-95% lifespan completed) WI-38 human diploid fibroblasts. We observed that, following stimulation with medium containing 10% fetal bovine serum (FBS), the translocation of PKC from the cytosol to the particulate compartment was less efficient in senescent WI-38 cells than in young cells. However, when PMA was added to the medium, the intracellular distribution of PKC activity in old cells became nearly identical to that observed in young cells. The inducibility of c- fos mRNA by serum addition, which is a protein kinase C-dependent event [64], was significantly amplified in the presence of PMA. Moreover, the duration of peak c- fos expression, after stimulation by FBS and PMA, increased in senescent cells as compared to young cells. Our results reveal that the normal signal transduction pathway is altered in senescent, slowly proliferating human fibroblasts and that it can be partially restored in the presence of the tumor promoter PMA.

Inhibition of Murine Sarcoma Cell Adherence to Polystyrene Substrata by Bacillus Calmette-Guérin: Evidence for Fibronectin-Mediated Direct Antitumor Activity of BCG

Bacillus Calmette-Guérin (BCG) inhibited adherence of S180 mouse sarcoma cells and WI38 human diploid fibroblasts to the polystyrene substratum of 24-well cluster dishes in a dose-dependent manner. This property was retained by washed or heat-killed bacilli, but not by the vaccine filtrate or by the spent bacterial culture medium. Adhesion of bacilli to nonadherent S180 cells was demonstrated by light and scanning electron microscopy, but was not seen after trypsinization of adherent cells, indicating that bacilli bind to cell-surface adhesins. Preincubation of bacilli with human fibronectin abolished their ability to inhibit S180 adherence, suggesting that the phenomenon may be mediated by interaction of bacilli with cell-surface fibronectin. Fibronectin pretreatment of the bacteria also decreased their inhibition of S180 tumor growth in vivo, indicating that this mechanism may be at least partly responsible for BCG vaccine's observed antineoplastic activity.