Increased tumor necrosis factor-alpha sensitivity of MCF-7 cells transfected with NAD(P)H:quinone reductase.

Evidence from a number of studies suggests that the mechanism by which tumor necrosis factor (TNF) kills transformed cells involves oxidative stress. NAD(P)H:(quinone acceptor) oxidoreductase (NQO1) is an antioxidant enzyme with particular relevance to cancer. The MCF-7 breast cancer cell line was stably transfected with rat NQO1 cDNA to determine whether increased NQO1 activity alters sensitivity to TNF-induced apoptosis. Five clones, with a range of NQO1 enzyme activities from 5- to 50-fold greater than the MCF-7 line, and two control transfectants were examined. Northern blot hybridization analyses and reverse transcription-PCR demonstrated that the increase in NQO1 activity in the transfectants was attributable to expression from the transfected rat sequence. Based on sulforhodamine B assays for the number of viable cells, the NQO1 clones showed increased sensitivity to EO9, an indoloquinone that undergoes bioactive reduction by NQO1. Viability studies also demonstrated that the NQO1 transfectants were significantly more sensitive to TNF than the control transfectants or MCF-7 parent. This increased sensitivity could not be explained by changes in superoxide dismutase or catalase activity or to increased sensitivity to oxidative stress in general, as assessed by response to hydrogen peroxide and paraquat treatment. Using dichlorodihydrofluorescein diacetate as a probe, we found that the NQO1 transfectants had no difference in baseline level of oxidative stress compared to the control cells but did exhibit greater intracellular oxidative stress after TNF treatment. We conclude that NQO1 can affect the TNF-mediated pathway to apoptosis.

Thioredoxin, a gene found overexpressed in human cancer, inhibits apoptosis in vitro and in vivo.

The redox protein thioredoxin plays an important role in controlling cancer cell growth through regulation of DNA synthesis and transcription factor activity. Thioredoxin is overexpressed by a number of human primary cancers and its expression is decreased during dexamethasone-induced apoptosis of mouse WEHI7.2 thymoma cells. We examined the ability of WEHI7.2 cells stably transfected with human thioredoxin cDNA showing increased levels of cytoplasmic thioredoxin to undergo apoptosis in vitro and in vivo. The cells were protected from apoptosis induced by dexamethasone, staurosporine, etoposide, and thapsigargin, but not by N-acetyl-sphingosine. When inoculated into severe combined immunodeficient mice, the trx-transfected cells formed tumors that showed increased growth compared to wild-type, as well as bcl-2-transfected, WEHI7.2 cells. The trx- and bcl-2-transfected cell tumors both showed less spontaneous apoptosis than tumors formed by the wild-type cells. Unlike tumors formed by the wild-type and bcl-2-transfected WEHI7.2 cells, trx-transfected cell tumors did not show growth inhibition upon treatment with dexamethasone. This study suggests that increased thioredoxin expression in human cancers may result in an increased tumor growth through inhibition of spontaneous apoptosis and a decrease in the sensitivity of the tumor to drug-induced apoptosis.

Catalase-overexpressing thymocytes are resistant to glucocorticoid-induced apoptosis and exhibit increased net tumor growth.

Glucocorticoids are used for the treatment of lymphoid neoplasms, taking advantage of the well-known ability of these compounds to cause apoptosis in lymphoid tissues. Previously, we have shown that dexamethasone, a synthetic glucocorticoid, causes a down-regulation of several antioxidant defense enzymes and proteins, including catalase and thioredoxin, concomitant with the induction of apoptosis in WEHI7.2 mouse thymoma cells. To test whether this down-regulation plays a critical role in the mechanism of steroid-induced apoptosis, WEHI7.2 cells were transfected with rat catalase. Two clones, expressing 1.4-fold and 2.0-fold higher catalase specific activity, respectively, when compared with vectoronly transfectants were selected for further study. An increase to 1.4-fold parental cell catalase activity delayed cell loss after dexamethasone treatment, whereas a 2.0-fold parental catalase activity prevented dexamethasone-induced cell loss for 48 h after treatment. Dexamethasone treatment of the WEHI7.2 cells stimulated a release of cytochrome c into the cytosol. Catalase-overexpressing cells showed a delay or lack of cytochrome c release from the mitochondria, which correlated temporally with the delay or prevention of cell loss in the culture after dexamethasone treatment. A decreased amount of cell death from WEHI7.2 cells overexpressing catalase was also seen in tumor xenografts in severe combined immunodeficient mice when compared with tumors from vector-only transfected cells. Similarly, thioredoxin-overexpressing WEHI7.2 cells, shown previously to be apoptosis resistant, showed decreased cell death in tumor xenografts. This resulted in larger tumors from cells overexpressing these proteins. Cell death in control transfectant tumor xenografts was primarily attributable to apoptosis. In contrast, the cell death we observed in tumors from thioredoxin- or catalase-overexpressing cells had a higher frequency of a nonapoptotic, nonnecrotic type of cell death termed para-apoptosis. These data suggest that: (a) oxidative stress plays a critical role in steroid-induced apoptosis prior to the commitment of the cells to undergo apoptosis; and (b) resistance to oxidative stress can contribute to tumor growth.

Transfection with human thioredoxin increases cell proliferation and a dominant-negative mutant thioredoxin reverses the transformed phenotype of human breast cancer cells.

Thioredoxin, a redox protein with growth factor activity that modulates the activity of several proteins important for cell growth, has been reported to be overexpressed in a number of human primary cancers. In the present study, the effects of stably transfecting mouse NIH 3T3 cells and MCF-7 human breast cancer cells with cDNA for wild-type human thioredoxin or a redox-inactive mutant thioredoxin, Cys32-->Ser32/Cys35-->Ser35 (C32S/C35S), on cell proliferation and transformed phenotype have been investigated. NIH 3T3 cells transfected with thioredoxin achieved increased saturation densities compared with vector alone-transfected cells, but were not transformed as assessed by tumor formation in immunodeficient mice. Thioredoxin-transfected MCF-7 cells showed unaltered monolayer growth on plastic surfaces compared with vector alone-transfected cells, but exhibited severalfold increased colony formation in soft agarose. Stable transfection of NIH 3T3 and MCF-7 cells with C32S/C35S resulted in inhibition of monolayer growth on plastic surfaces, and up to 73% inhibition of colony formation by MCF-7 cells in soft agarose. When inoculated into immunodeficient mice, thioredoxin-transfected MCF-7 cells formed tumors, although with a 38-57% growth rate compared with vector alone-transfected cells, whereas tumor formation by C32S/C35S-transfected MCF-7 cells was almost completely inhibited. The results of the study suggest that thioredoxin plays an important role in the growth and transformed phenotype of some human cancers. The inhibition of tumor cell growth by the dominant-negative redox-inactive mutant thioredoxin suggests that thioredoxin could be a novel target for the development of drugs to treat human cancer.

Modulation of the antioxidant defence as a factor in apoptosis.

This review focuses on evidence that oxidative stress during apoptosis is controlled, at least in part, by modulating cellular antioxidant defences. Evidence is presented from studies of apoptosis induced by glucocorticoids, HIV-1 infection and tumour necrosis factor-alpha. Glucocorticoid treatment of murine lymphocyte cell lines leads to the down-regulation of primary antioxidant defence enzymes, including catalase, superoxide dismutases, thioredoxin and DT-diaphorase. Following HIV-1 infection, disturbances in glutathione metabolism are seen, and decreased antioxidant enzyme activities have been reported for HIV-1-infected cell lines. The viral protein Tat may mediate these effects. Cellular resistance to apoptosis induced by tumour necrosis factor-alpha is modulated by the expression of manganese superoxide dismutase or Bcl-2. The loss of antioxidant defences is predicted to lead to oxidative stress, which could contribute to the mechanism of apoptosis through an effect on redox-sensitive transcription factors, calcium homeostasis or cysteine proteases.

Thymocytes selected for resistance to hydrogen peroxide show altered antioxidant enzyme profiles and resistance to dexamethasone-induced apoptosis.

Treatment of WEHI7.2 cells, a mouse thymoma-derived cell line, with dexamethasone, a synthetic glucocorticoid, causes the cells to undergo apoptosis. Previous work has shown that treatment of WEHI7.2 cells with dexamethasone results in a downregulation of antioxidant defense enzymes, suggesting that increased oxidative stress may play a role in glucocorticoid-induced apoptosis. To test whether resistance to oxidative stress causes resistance to dexamethasone-induced apoptosis, WEHI7.2 cell variants selected for resistance to 50, 100 and 200 microM H(2)O(2) were developed. Resistance to H(2)O(2) is accompanied by increased antioxidant enzyme activity, resistance to other oxidants and a delayed loss of viable cells after dexamethasone treatment. In the 200 microM H(2)O(2)-resistant cell variant the delay in cell loss is correlated with delayed release of cytochrome c from the mitochondria into the cytosol. This suggests that reactive oxygen species play a role in a signaling event during steroid-mediated apoptosis in lymphocytes.

Downregulation of the antioxidant defence during glucocorticoid-mediated apoptosis.

Recent studies implicate oxidative stress in the mechanism of apoptosis. We have examined the expression of genes, whose products counteract oxidative stress, during glucocorticoid-mediated apoptosis of a murine thymoma-derived cell line. Using Northern blot hybridisation analyses, we observed a progressive decline over a 24 h period in the transcript levels for catalase, manganese superoxide dismutase, copper, zinc-superoxide dismutase, DT-diaphorase and thioredoxin. The changes were first seen within 2-8 h of the addition of the hormone which is well in advance of appreciable apoptosis. Using Western blot hybridisation analyses we found that a dexamethasone-mediated increase in glutathione S-transferase message level was followed closely by an increase in glutathione S-transferase mu class protein and a 20% decrease in reduced glutathione levels. Our findings suggest that the downregulation of cellular oxidant defense enzymes with a consequent increase in oxidant damage could contribute to the molecular mechanism of apoptosis.

Decreased antioxidant defence and increased oxidant stress during dexamethasone-induced apoptosis: bcl-2 prevents the loss of antioxidant enzyme activity.

When the WEHI7.2 mouse lymphoid cell line was treated with dexamethasone to induce apoptosis the activities and transcript levels of the antioxidant defence enzymes catalase, superoxide dismutase (SOD) and DT-diaphorase exhibited a progressive decrease over 48 hours. Catalase activity was maintained and total SOD and DT-diaphorase activity showed smaller decreases following dexamethasone treatment of WEHI7.2 cells transfected with the bcl-2 oncogene, which protects the cells against apoptosis. Treatment of wild-type WEHI7.2 and bcl-2 transfected cells with a catalase inhibitor, aminotriazole, was not sufficient to induce apoptosis. Antioxidants, including bovine liver catalase, bovine erythrocyte CuZn-SOD, sodium selenite and Trolox, a water soluble vitamin E analogue, as well as hypoxia, inhibited dexamethasone-induced apoptosis. These results suggest that oxidant stress due to the decreased activity of antioxidant defence enzymes may play a role in dexamethasone-mediated lymphoid cell apoptosis and that bcl-2 may prevent apoptosis by maintaining the level of critical antioxidant defence mechanisms, which include catalase.

Isolation and characterization of transcripts induced by androgen withdrawal and apoptotic cell death in the rat ventral prostate.

A variety of stimuli have been identified which initiate transcription-dependent programmed cell death (apoptosis) in specific target cells. Since the withdrawal of androgens induces regression and apoptosis in rat ventral prostate (RVP) epithelial cells, and it is known that the androgen receptor is a transcriptional regulator, we used subtraction cDNA cloning to isolate differentially expressed transcripts from the RVP of androgen ablated rats. In addition to sulfated glycoprotein-2 and glutathione S-transferase (GST), which had been previously described, several other transcripts were found to be elevated 3- to 8-fold in the regressing RVP. DNA sequencing revealed that two of these cDNA clones encode matrix carboxyglutamic acid and gamma-actin, respectively. A third cDNA contained novel sequence information and was named RVP.1. The RVP.1 transcript is expressed at very low levels in the RVP and epididymis of normal adult rats (less than 0.01% of the total mRNA) and is undetectable in other tissues, such as kidney, liver, and muscle. RVP.1 encodes a putative 280-amino acid protein, which shares no significant homology with previously described protein functional domains. We examined the expression of these transcripts in serum-starved NIH 3T3 cells to determine whether any of them are elevated in cells that are growth arrested. It was found that only GST mRNA levels are increased under these conditions. These data may suggest that induction of some genes, such as RVP.1, could be associated with apoptosis, whereas other transcripts, such as GST, may be up-regulated in response to altered rates of cellular metabolism.

Transcriptional analyses of steroid-regulated gene networks.

It has been proposed that cell-specific responses to steroid action are the result of coordinate expression of steroid gene networks. Using three different cell systems, we have performed transcriptional analyses to determine if the observed hormone-induced alterations in gene expression are consistent with a limited number of potential target genes in any one cell type. Our results indicate that greater than 95% of the transcripts in dexamethasone-treated rat hepatoma (HTC), or mouse lymphoma (WEH17) cells, are similar to the mRNAs in untreated cells based on subtraction hybridization. In addition, we find that although the castration-induced expression of androgen-regulated transcripts in the rat ventral prostate (RVP) is significantly different between normal and castrated rats (19%), the majority of these mRNAs are accounted for by the over abundance of sulfated glycoprotein-2 sequences. Specifically, analysis of an RVP subtracted cDNA library revealed that sulfated glycoprotein-2 mRNA masked the presence of less abundant differentially expressed sequences, confirming that the actual size of the RVP androgen gene network is small. We conclude that steroid-mediated changes in transcription accurately reflect the expression of a few cell-specific target genes, and thus support the model of steroid gene networks. The potential to characterize key elements which determine both the time course and magnitude of cell-specific hormone responses is discussed.

Antioxidant defenses in the TNF-treated MCF-7 cells: selective increase in MnSOD.

Oxidative stress has been implicated in the mechanism of tumor necrosis factor-alpha (TNF)-induced apoptosis, raising a question about the status of antioxidant defenses in TNF-sensitive cells. Antioxidant defenses were examined in MCF-7 cells after treatment with TNF. Cell morphology and DNA fragmentation assays were used to confirm increased apoptosis as a result of TNF treatment. The expression and activity of antioxidant defenses were assessed using Northern blot hybridization analyses and biochemical assays, respectively. Five- and ten-fold increases in manganese superoxide dismutase (MnSOD) mRNA were measured after one and five days of TNF treatment, respectively. The expression of copper,zinc superoxide dismutase, catalase or thioredoxin was not altered. An approximate five-fold increase in MnSOD activity followed the change in gene expression, but no difference in the activity of catalase or glutathione peroxidase was seen. Thus, increased MnSOD activity was not accompanied by an increase in other antioxidant defenses and in particular, H2O2-scavenging enzymes. MnSOD has previously been shown to afford protection against TNF-mediated cytotoxicity. The observed lack of increased peroxidase activity is consistent with mitochondrially-generated superoxide anion radical contributing to the mechanism of TNF-induced apoptosis.

Attenuation of catalase activity in the malignant phenotype plays a functional role in an in vitro model for tumor progression.

We have developed an in vitro model to study the molecular mechanisms of tumor progression. Using repeated treatments with ionizing radiation or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), we caused malignant progression of a papilloma producing mouse keratinocyte cell line, 308 cells. In a previous study we have shown that the malignant variants of 308 cells have elevated reactive oxygen species (ROS) levels, and have established a functional role for the pro-oxidant state in the progressed phenotype (Carcinogenesis 20 (1999) 2063). In this study, we have evaluated the status of intracellular defense mechanisms for ROS scavenging in the progressed phenotype to identify sources that contribute to their pro-oxidant state. Our results demonstrate that a reduction in several anti-oxidant defense mechanisms, including catalase and glutathione S-transferase mu, correlates with the emergence of the malignant phenotype. We provide evidence that attenuation of catalase activity may play a functional role in the malignant progression of mouse keratinocytes.

Induction of oxidative stress and apoptosis in myeloma cells by the aziridine-containing agent imexon.

Imexon is an iminopyrrolidone derivative that has selective antitumor activity in multiple myeloma. The exact mechanism of imexon action is unknown. In human 8226 myeloma cells, the cytotoxicity of imexon was schedule-dependent, and long exposures (> or = 48 hr) to low concentrations of imexon were most effective at inducing cytotoxicity. Our data suggest that imexon does not affect DNA, but it can alkylate thiols by binding to the sulfhydryl group. We have also demonstrated by HPLC studies that in human 8226 myeloma cells, imexon depletes cellular stores of cysteine and glutathione. Oxidative stress in 8226 cells exposed to imexon was detected by immunohistochemical staining with a monoclonal antibody to 8-hydroxydeoxyguanosine (8-OHdG), followed by confocal microscopy. These images showed increased levels of 8-OHdG in the cytoplasm of cells treated with different concentrations of imexon at 8, 16, and 48 hr. Interestingly, 8-OHdG staining was not observed in the nuclei of imexon-treated cells, in contrast to the diffuse staining seen with t-butyl hydroperoxide. Myeloma cells exposed to imexon showed classic morphologic features of apoptosis upon electron microscopy, and increased levels of phosphatidylserine exposure, detected as Annexin-V binding, on the cell surface. To prevent depletion of thiols, 8226 myeloma cells exposed to imexon were treated with N-acetylcysteine (NAC). Simultaneous, as well as sequential, treatment with NAC before imexon exposure resulted in protection of myeloma cells against imexon-induced cytotoxicity. Conversely, the glutathione synthesis inhibitor buthionine sulfoximine increased imexon cytotoxicity. These data suggest that imexon perturbs cellular thiols and induces oxidative stress leading to apoptosis in human myeloma cells.

Modulation of antioxidant defenses during apoptosis.

Understanding the fundamental mechanism of apoptosis is crucial to developing therapeutic strategies for controlling apoptosis in diseased tissues. We are using model systems with relevance to cancer treatment to investigate the mechanism of apoptosis. Subtraction hybridization cloning was used to identify transcripts present at higher levels in regressing vs. normal prostate; these may be important for apoptosis. One of the genes cloned from regressing prostate is also upregulated in the murine W7.2 lymphocyte cell line induced to undergo apoptosis by treatment with the synthetic glucocorticoid, dexamethasone. This gene encodes a mu class glutathione S-transferase (EC 2.5.1.18), a protein that can protect the cell against oxidative stress by repairing oxidized lipids, proteins, and DNA. Glutathione S-transferase expression does not increase with dexamethasone treatment of lymphocyte cell lines expressing nonfunctional glucocorticoid receptors or a mutation in the apoptotic pathway. Other antioxidant defenses, including catalase (EC 1.11.1.6) and superoxide dismutase (EC 1.15.1.1), decline following dexamethasone treatment of W7.2 cells. Overexpression of the bcl-2 oncogene protects these cells against dexamethasone-mediated apoptosis and prevents the decrease in antioxidant enzyme activity. These findings support the hypothesis that control of the cellular redox state is important to the mechanism of glucocorticoid-mediated lymphocyte apoptosis. Another model system we are using is tumor necrosis factor-alpha treatment of MCF-7 human breast cancer cells. Our preliminary results suggest that, in this system, activation of nuclear factor-kappa B and increased expression of manganese superoxide dismutase may afford protection from apoptosis.

Helix hand fidelity in Bacillus subtilis macrofibers after spheroplast regeneration.

Left- and right-handed Bacillus subtilis macrofibers produced by strains FJ7 and C6D were converted to spheroplasts. Intact cells were regenerated and macrofibers were produced under conditions conducive for production of left- and right-handed structures. The resulting helix hand phenotypes always corresponded to those expected on the basis of the parental genotype.

Changes in phosphate metabolism in thymoma cells suggest mechanisms for resistance to dexamethasone-induced apoptosis. A 31P NMR spectroscopic study of cell extracts.

Treatment of the mouse thymoma-derived WEHI7.2 cell line with dexamethasone, a synthetic glucocorticoid, causes the cells to undergo apoptosis. Previous studies have shown that WEHI7.2 cell variants with an increased antioxidant defense exhibit increased resistance to dexamethasone-induced apoptosis, suggesting that oxidative stress may play a role in glucocorticoid-induced apoptosis. In this work we compared metabolic profiles of WEHI7.2 parental cells with those of WEHI7.2 variants with an increased antioxidant defense or overexpressing bcl-2, to determine whether bolstering the antioxidant defense results in altered metabolic parameters that could translate into increased resistance to dexamethasone-induced apoptosis. WEHI7.2 parental cells and cells overexpressing catalase, thioredoxin or bcl-2, or selected for resistance to 200 micro M H(2)O(2) were cultured in low-glucose DMEM medium supplemented with 10% calf serum, and extracted using chloroform-methanol-water (1:1:1). Metabolites contained in the aqueous and organic phases of the extracts were processed separately and subjected to high-resolution (31)P NMR spectroscopy. In most of the steroid-resistant variants, ATP levels and energetic status were decreased compared with the steroid-sensitive parental cell line, while the concentrations of hexose and triose phosphates were increased. Furthermore, the ratio of choline-containing phospholipids to ethanolamine-containing phospholipids was generally reduced in steroid-resistant cells. Phosphatidylethanolamine and its derivatives contain a higher amount of polyunsaturated fatty acids (PUFA) than the choline-containing analogs, and PUFA are readily oxidized by reactive oxygen species. Therefore, an increased initial amount of phosphatidylethanolamine may increase the 'buffering capacity' of this antioxidant and may thus contribute to the steroid resistance of WEHI7.2 variants.

Elevated glutathione S-transferase gene expression is an early event during steroid-induced lymphocyte apoptosis.

Based on the finding that glutathione S-transferase Yb1 (GST) gene expression is elevated in the regressing prostate of androgen-ablated rats, we analyzed GST transcript levels during steroid-induced lymphocyte cell death. It was found that GST gene expression was induced in steroid-sensitive cells within 4 hr of dexamethasone treatment, required functional glucocorticoid receptor, and was dose-dependent with regard to hormone. GST expression was not induced in an apoptosis-defective variant that contained normal levels of functional receptor, indicating that GST up-regulation was the result of secondary events that occur during steroid-mediated apoptosis. Using the calcium ionophore A23817 to induce lymphocyte cell death, GST RNA levels were increased in both steroid-sensitive and steroid-resistant cell lines, supporting the conclusion that elevated GST expression was the result of cellular processes associated with apoptosis, rather than a direct consequence of steroid-mediated transcriptional control. The cells were also treated with dibutyryl cAMP to cause cell death; however, this mode of killing did not result in GST up-regulation. Taken together, these results suggest that GST induction in dexamethasone-treated T-lymphocytes occurs early in the steroid-regulated apoptotic pathway and that this may be a marker of calcium-stimulated cell death. Based on the known function of GST as an antioxidant defense enzyme and its transcriptional regulation by reactive oxygen intermediates, we propose that the gene product of a primary GR target gene(s) directly or indirectly effects the redox state of the cell. Thus activation of GST gene expression in apoptotic lymphocytes is likely a indicator of oxidative stress, rather than a required step in the pathway.

Induction of mitochondrial changes in myeloma cells by imexon.

Imexon is a cyanoaziridine derivative that has antitumor activity in multiple myeloma. Previous studies have shown that imexon induces oxidative stress and apoptosis in the RPMI 8226 myeloma cell line. This study reports that imexon has cytotoxic activity in other malignant cell lines including NCI-H929 myeloma cells and NB-4 acute promyelocytic leukemia cells, whereas normal lymphocytes and U266 myeloma cells are substantially less sensitive. Flow cytometric experiments have shown that imexon treatment is associated with the formation of reactive oxygen species (ROS) and the loss of mitochondrial membrane potential (Deltapsi(m)) in imexon-sensitive myeloma cell lines and NB-4 cells. In contrast, reduction of Deltapsi(m) and increased levels of ROS were not observed in imexon-resistant U266 cells. Treatment of imexon-sensitive RPMI 8226 cells with the antioxidant N-acetyl-L-cysteine (NAC) protects cells against these effects of imexon. Mitochondrial swelling was observed by electron microscopy in RPMI 8226 myeloma cells treated with 180 microM imexon as early as 4 hours. Damage to mitochondrial DNA was detected by a semiquantitative polymerase chain reaction assay in imexon-treated RPMI 8226 cells; however, nuclear DNA was not affected. Finally, partial protection of RPMI 8226 cells against the imexon effects was achieved by treatment with theonyltrifluoroacetone, an inhibitor of superoxide production at mitochondrial complex II. These changes are consistent with mitochondrial oxidation and apoptotic signaling as mediators of the growth inhibitory effects of imexon. Interestingly, oxidative damage and decrease of Deltapsi(m) induced by imexon highly correlates with sensitivity to imexon in several myeloma cell lines and an acute promyelocytic leukemia cell line. (Blood. 2001;97:3544-3551)

Factors contributing to helical shape determination and maintenance in Bacillus subtilis macrofibres.

Bacillus subtilis, normally a rod-shaped organism, can grow in the form of a helix with pitch ranging over a spectrum from tight right-handed to tight left-handed depending upon the growth environment and genetic composition of the strain. Five factors have been identified which contribute either to the helical shape deformation or its maintenance: 1) a biomechanical component involving blocked rotation during growth; 2) cell wall polymer conformation; 3) a protein(s) concerned with the left-hand form produced at high temperature; 4) electrostatic aspects of the cell wall; and 5) water, as it affects the mechanical properties of cell walls and the structure of cell wall polymers. The findings are compatible with a model in which the cell wall polymers are inserted in helical orientation along the cylindrical portion of the cell during growth.