Differential expression of survivin splice isoforms in medulloblastomas.

Survivin, a member of the inhibitor of apoptosis protein family, is implicated in the dysregulation of apoptosis in human cancers. Survivin and survivin-deltaEx3, one of its two alternatively spliced isoforms, confer anti-apoptotic activities in human tumours, while survivin-2B antagonizes such anti-apoptotic properties. The current study was undertaken to examine the mRNA expression of survivin isoforms and their correlation with clinical staging and outcome in 20 medulloblastoma (MB) tumours, three MB cell lines and normal brain tissues (a foetal and an adult cerebellum) by densitometry scanning of 32p-dCTP incorporated reverse transcription polymerase chain reaction (RT-PCR) products and quantitative real-time PCR. Our results showed that the normal adult brain only expressed low levels of survivin-deltaEx3 mRNA, while the foetal brain expressed all three isoforms, with wild-type survivin as the dominant transcript. All three survivin isoforms were detected in all the MB cell lines and tumours analysed. Immunohistochemical staining also demonstrated survivin protein expressions in all five paraffin-embedded MBs, with predominant nuclear localization. Although overexpressions of survivin were not associated with the presence of metastatic MB or tumour histological subtypes, elevated expressions of survivin-deltaEx3 were significantly associated with progressive/recurrent tumours (P-value = 0.024). Our data demonstrated that overexpression of survivin mRNA is a common feature in MBs, may contribute to their anti-apoptosis properties and clinical behaviours, and predicts a poor clinical outcome, independent of clinical staging or tumour histology.

NAD(P)H:quinone oxidoreductase 1 deficiency increases susceptibility to benzo(a)pyrene-induced mouse skin carcinogenesis.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoprotein that catalyzes the metabolic detoxification of quinones and their derivatives. This protects cells against quinone-induced oxidative stress, cytotoxicity, and mutagenicity. C57BL6 NQO1-/- mice, deficient in NQO1 RNA and protein, were generated in our laboratory. To investigate the role of NQO1 in chemical carcinogenesis, the dorsal skin of NQO1-deficient (NQO1-/-) and wild-type (NQO1+/+) mice were treated with a single dose of benzo(a)pyrene, followed by twice weekly applications of phorbol-12-myristate-13-acetate. The NQO1-/- mice showed a much higher frequency of skin tumor development when compared with their wild-type littermates. Interestingly, the male NQO1-/- mice were slower to develop skin tumors than their NQO1-/- female littermates. Histological analysis of the NQO1-/- tumors showed proliferative activity. These results demonstrate that NQO1 acts as an endogenous factor in protection against benzo(a)pyrene carcinogenicity.

The silver reaction of nucleolar proteins in the main structural compartments of ring-shaped nucleoli in smear preparations.

The present study was undertaken to provide more information on the conditions which result in preferential silver staining of the main nucleolar structural compartments using silver stainable proteins as their markers at the light microscopic level. For this study the mostly used method in cytology and pathology in which the nucleolar silver-positive structures are "developed" with the colloidal developer (Howell and Black, 1980; Ploton et al., 1986) was selected as silver reaction. Ring-shaped nucleoli of mature human lymphocytes represent a convenient model for such a study because they consist of one large fibrillar center, adjacent nucleolar regions with dense fibrillar components and the nucleolar peripheral shell with dense granular components. All these nucleolar compartments are known to possess characteristic silver stainable proteins. The results demonstrated that proteins of the fibrillar center and possibly adjacent nucleolar regions reacted preferentially with silver after a relatively long fixation with formaldehyde or methanol in unwashed specimens before the silver reaction. In contrast, the preferential staining of proteins in the nucleolar peripheral shell with silver was achieved after the fixation with acidified methanol or ethanol as well as after short fixation with formaldehyde vapors. In addition, the commonly used fixation before the silver reaction are not necessary and may be omitted for the visualization of all silver stainable proteins present in the fibrillar center as well as in the adjacent nucleolar regions and the nucleolar peripheral shell. In addition, similar results were achieved for the simultaneous visualization of proteins in the fibrillar center and nucleolar peripheral shell after fixation with ethanol.

The 58-kDa microspherule protein (MSP58), a nucleolar protein, interacts with nucleolar protein p120.

Protein p120 is a proliferation-related nucleolar protein which is detectable early in the G1 phase of the cell cycle and peaks early in the S phase. Most human malignant tumors contain much higher levels of protein p120 than normal resting cells. To identify p120-associated protein(s), a yeast two-hybrid screen was carried out using protein p120 as the bait. Two positive clones encoded portions of a novel protein, designated microspherule protein 58 kDa (MSP58). MSP58 mRNA is 1.9 kb and encodes an approximately 58-kDa polypeptide of 462 amino acids as shown by Western blotting of HeLa nucleolar proteins. The mouse MSP58 homolog has 97% amino acid similarity to human MSP58, but no MSP58 homolog was found in the yeast genome. The MSP58 N-terminal region contains serine-rich clusters and its C-terminal region has a coiled-coil domain. In insect Sf9 cells, recombinant p120 and MSP58 proteins associated with each other, confirming the results of the yeast two-hybrid assay. Deletion mutations revealed that the binding of MSP58 to p120 required a previously unrecognized coiled-coil domain within the N-terminal region of p120 and the C-terminal region of MSP58 protein. Immunofluorescence indicated that the MSP58 protein is localized in microspherules in the nucleolus. Anti-MSP58 Ig labeled nucleolar 'caps' when HeLa cells were treated with actinomycin D. When the MSP58 protein was overexpressed in COS-7 cells, the nucleolus became irregularly enlarged, which suggests that MSP58 may affect the size and shape of the nucleolus.

Green fluorescent protein tag for studies of drug-induced translocation of nucleolar protein RH-II/Gu.

We have constructed a human osteogenic sarcoma cell line, U-2 OS/GFP-Gu, that expresses nucleolar RNA helicase RH-II/Gu tagged with green fluorescent protein (GFP). The presence of a GFP tag does not inhibit RNA helicase, RNA folding and ATPase activities of RH-II/Gu protein. The derived cell line responds to cytotoxic agents like the parental cell line U-2 OS. In the presence of either actinomycin D or toyocamycin, the GFP-RH-II/Gu fusion protein translocates from the nucleolus to the nucleoplasm in the same way as the translocation of endogenous RH-II/Gu. The drug-induced translocation of GFP-RH-II/Gu is easily monitored by direct observation of live cells in vivo. This cell line can be used to screen cytotoxic drugs and to study the mechanisms of drug-induced translocation of RH-II/Gu. The cellular localization of RH-II/Gu during the cell cycle-dependent formation of the nucleolus is readily monitored. Real-time results are obtained more quickly without the disadvantages associated with cell fixation and immunofluorescence-based staining.

Effects of cytotoxic drugs on translocation of nucleolar RNA helicase RH-II/Gu.

Some cytotoxic drugs cause translocation of nucleophosmin/B23 and other nucleolar proteins to the nucleoplasm. The present study shows that these drugs caused a similar translocation of RH-II/Gu, a nucleolar RNA helicase. Other nucleolar proteins including p120, UBF, RNA polymerase I large subunit, fibrillarin, p40, and Ren-1 did not translocate. A 2-h treatment of MCF-7 breast cancer cells with 0.008 or 0.16 microM actinomycin D resulted in translocation of RH-II/Gu to the nucleoplasm; these effects were not reversed by 100 microM guanosine. The effects of 0.008 microM actinomycin D, but not 0.16 microM actinomycin D, on the translocation of RH-II/Gu were reversed when the drug was removed. However, the effects of 0.008 or 0.16 microM actinomycin D on the translocation of nucleophosmin/B23 were not reversible. The translocation effects of 50 microM toyocamycin on RH-II/Gu were reversed when the drug was replaced with fresh medium. RH-II/Gu mostly relocalized to the nucleoli within 15 min after toyocamycin was withdrawn; only partial relocalization of nucleophosmin/B23 occurred 40 h after removal of the drug. The effects of toyocamycin were not blocked by 100 microM guanosine. Mycophenolic acid (50 microM, 2-h treatment) caused partial translocation of RH-II/Gu; this effect was slowly reversed upon drug removal and was inhibited by 100 microM guanosine, in a manner similar to the effects of mycophenolic acid on the localization of nucleophosmin/B23. This study shows similarities and differences in the drug-induced translocation and relocalization of RH-II/Gu and nucleophosmin/B23. Analysis of translocation of specific nucleolar proteins may offer a quantitative approach to assessment of potency and duration of effects of cytotoxic agents.

Cloning and characterization of a new silver-stainable protein SSP29, a member of the LRR family.

Silver-stainable proteins (SSPs) are aspartic acid-rich nuclear proteins which are silver stained under very specific conditions. Using a degenerate oligodeoxyncleotide probe which codes for acidic amino acid residues, a cDNA for a new SSP, referred to as SSP29, has been isolated. The cDNA-derived amino acid sequence shows SSP29 has a molecular mass of 29 kDa, leucine-rich repeats (LRR) near the NH2-terminal region and acidic clusters at the COOH-terminal portion, indicating that SSP29 is also a member of the LRR subfamily of acidic proteins which have been shown to be involved in antigen-mediated cellular responses, leukemogenesis and differentiation. SSP29 can be stained by Ag-NOR staining. SSP29 is expressed in all human tissues and cell lines tested, localized to nucleoplasm and translocated partially to the nucleoli after heat shock. Its interaction with RNA polymerase I suggests that SSP29 may participate in signal transduction that directs nucleolar activities by regulating ribosomal RNA biosynthesis.

Cloning and characterization of Gu/RH-II binding protein.

Gu/RNA helicase II (Gu/RH-II) is the first reported mammalian nucleolar RNA helicase that is a member of the D-E-A-D (Asp-Glu-Ala-Asp) box family of proteins. It has an ATP-dependent RNA unwinding (helicase) activity and a separate RNA folding activity (introduction of intramolecular secondary structure into single-stranded RNA). To determine which proteins may bind to Gu/RH-II, a yeast two-hybrid system was used. A cDNA which encoded a protein, called Gu/RH-II binding protein or GBP, was isolated and sequenced. The GBP protein is localized to the nucleus in speckled or diffuse nucleoplasmic patterns. The GBP mRNA level is highest in testis, 9- to 49-fold greater than other tissues. When GBP interacts with Gu/RH-II, proteolytic cleavage of Gu/RH-II occurs; the amino-terminal portion of Gu/RH-II is critical for this proteolysis.

A nucleolar RNA helicase recognized by autoimmune antibodies from a patient with watermelon stomach disease.

Watermelon stomach is characterized by prominent stripes of ectatic vascular tissue in the stomach similar to stripes on a watermelon; in patients with this disorder chronic gastrointestinal bleeding occurs and approximately half of these patients have associated autoimmune disorders. In the serum of one patient, an antinucleolar antibody titer of 1:25 600 was found; the antibodies specifically recognized an approximately 100 kDa nucleolar protein, which we referred to as the 'Gu' protein. Its cDNA was cloned and sequenced. The Gu protein is a member of a new subgroup of RNA helicases, the DEXD box family. Gu protein fused with glutathione S-transferase contains ATP-dependent RNA helicase activity which preferably translocates in the 5'-->3' direction. Its RNA folding activity, RNA-dependent ATPase and dATPase activities, and its translocation direction are similar to those of RNA helicase II [Flores-Rozas, H. and Hurwitz, J. (1993) J. Biol. Chem. 268, 21372-21383]. Sequencing of 209 amino acids of RNA helicase II peptides showed 96.7% identity with the cDNA-derived amino acid sequence of the Gu protein. The precise biological roles of this RNA helicase in the biogenesis of ribosomal RNA and the pathogenesis of watermelon disease and autoimmune disorder require further study.

Combinatorial effects of monoclonal anti-p120 antibody (MAbp120), liposomes and hyperthermia on MCF-7 and LOX tumor cell lines.

The human nucleolar protein p120 is highly expressed in human cancers. Its high expression in breast cancer correlates with a poor prognosis, and its overexpression in 3T3 mouse fibroblasts causes malignant transformation. This study reports that a combination of monoclonal anti-p 120 antibody (MAbp120), liposomes (Lipo), and hyperthermia (HT) resulted in enhanced antitumor effects in cultured human breast adenocarcinoma (MCF-7) and human amelanotic melanoma (LOX) cells. Monoclonal antibody uptake and intracellular localization of the protein p120 were monitored by double labeling indirect immunofluorescence. Cell growth inhibition by the combination of MAbp120 + Lipo + HT was 65% for MCF-7 cells and 96% for LOX cells. When tested on LOX cells, monoclonal antibodies (MAbB23, MAbC23) to other nucleolar proteins (B23, C23) produced only slight cytotoxicity with similar treatment protocols.

Apoptosis in human tumor cells following treatment with p120 antisense oligodeoxynucleotide ISIS 3466.

Previously, we reported that treatment of LOX cells in vitro with phosphorothioate oligonucleotide ISIS 3466 (antisense to the human nucleolar protein p120-FB2) produced a 70% cell kill and morphological changes including nucleolar unravelling, chromatin condensation and fragmentation, and a reduction in mitotic figures consistent with apoptosis. This report shows that HeLa cells treated with ISIS 3466 also developed apoptosis: nucleosomal ladders were found when the DNA from the treated HeLa cells was extracted and run on agarose gels. The morphological changes consistent with apoptosis were found more frequently in the floating cells than in the attached cells. The percentages of floating cells and attached cells were indicators of the toxicity of the different oligonucleotides studied. Of these, oligonucleotide ISIS 3466 produced the highest percent of floating cells (78.4%). Treatment of HeLa cells with other oligonucleotides produced fewer floating cells, and the characteristic nucleosomal ladder was not found following DNA extraction.

Identification of the nuclear and nucleolar localization signals of the protein p120. Interaction with translocation protein B23.

The human p120 nucleolar protein is a cell cycle-related protein that peaks during the S phase and has been shown to be associated with a beaded fibrillar structure. To study domains responsible for the nucleolar localization of protein p120, initially deletion mutants were made that defined sequences containing the localization signals; then, fusion genes that were composed of segments of the p120 molecule joined to the N-terminal end of the Escherichia coli beta-galactosidase were constructed. In the absence of the localization signals the beta-galactosidase remained in the cytoplasm. When the identified nuclear localization signal containing the amino acid sequence 99-110 (NAPRGKKRPAPG) was fused to the beta-galactosidase, the protein localized to the nucleus. When only the identified nucleolar localization signal containing the amino acid sequence 40-57 (SKRLSSRARKRAAKRRLG) was fused to the beta-galactosidase, the fusion protein remained in the cytoplasm. When both the nuclear and nucleolar localization signals were fused to the beta-galactosidase it localized predominantly to the nucleolus. Nucleolar protein B23, a putative "shuttle protein," bound to amino acid sequence 24-56 of protein p120. Deletion analysis showed that amino acids 187-215 of protein B23 bound to protein p120. The results suggest that protein B23 may be part of the mechanism of protein targeting to the nucleolus.

Pharmacokinetics, tissue distribution, and stability of antisense oligodeoxynucleotide phosphorothioate ISIS 3466 in mice.

Phosphorothioate oligonucleotides have a potential as therapeutic agents. The pharmacokinetics, tissue distribution, stability, and cellular uptake by LOX ascites tumor of p120 antisense phosphorothioate oligonucleotide, ISIS 3466, were studied in vivo. The oligonucleotide, which was quickly cleared from the circulation in the normal mice after IV injection, was readily absorbed into the systemic circulation from the peritoneum. The oligonucleotide was found in most tissues 48 h after IP administration. The highest concentrations were in kidney and liver, but the brain had a very low concentration. The phosphorothioate oligonucleotide was intact even after 48 h. When the oligonucleotide was complexed with cationic lipid DOTMA, the DOTMA did not affect the oligonucleotide uptake or tissue distribution in normal mice. However, DOTMA significantly increased the oligonucleotide cellular uptake (4-10 times) in LOX ascites tumors in an IP/IP model. These results indicate that the phosphorothioate oligonucleotide is stable, has favourable kinetics for use as an therapeutic agent, and that DOTMA could be useful in local delivery of the oligonucleotide in vivo.

Nucleolar and nuclear aberrations in human lox tumor cells following treatment with p120 antisense oligonucleotide ISIS-3466.

Previous reports from this laboratory have shown marked cytocidal effects of the ISIS-3466 antisense phosphorothioate oligodeoxynucleotide to the human nucleolar protein p120 on human cancer cell lines in vitro and inhibition of tumor growth in vivo in an i.p/i.p. LOX cell model (L. Perlaky et al. Anti-Cancer Drug Design 8:3-14, 1993). In this study, light and fluorescence microscopy showed that the number of LOX cells in mitosis decreased by 50% after incubation for 4 h in 0.2-0.4 microM antisense oligonucleotide; a 70% reduction in cell number was found from 8-72 h post-treatment. In addition, marked unravelling of nucleolar structures and chromatin fragmentation was found after a 4-h incubation. The nucleolar unravelling occurred in varying degrees ranging from partial unfolding to almost complete separation of the strands of nucleolar residues. Twenty four hours post-treatment, immunofluorescence staining with the anti-p120 monoclonal antibody showed reduced nucleolar protein p120 and translocation of the p120 protein from the nucleoli to the nucleoplasm. Analysis of the mechanisms of the nucleolar unravelling and inhibition of mitosis will provide further understanding of the cytocidal effects of the ISIS-3466 antisense oligonucleotide.

The effect of antisense p120 construct on p120 expression and cell proliferation in human breast cancer MCF-7 cells.

Malignant transformation of NIH3T3 cells was observed by transfection with the pSVX vector containing a sense human p120 cDNA construct (pSVX120). Subsequent transfection of these transformed cells with a dexamethasone inducible antisense p120 construct (pMSG021) markedly reduced the expression of human p120 and the growth rate of these transformed cells (Perklaky et al., Cancer Res., (1992) 52, 428-436). In the present study, a human breast cancer cell line (MCF-7) which expresses the p120 protein was transfected by electroporation with a pSVX plasmid-construct containing the antisense p120 cDNA (pSVX021). Clones containing the pSVX021 construct were selected and analyzed for expression of p120 mRNA, protein and growth characteristics. The expression of the p120 protein was inhibited by 44% in the antisense-transfected MCF-7pSVX021 cells; a 56% inhibition of cell-growth and a reduced colony formation in soft agarose were also observed. The growth of MCF-7 cells transfected with the p120 antisense construct was reduced by 93% in nude mice.

Growth inhibition of human tumor cell lines by antisense oligonucleotides designed to inhibit p120 expression.

The human nucleolar antigen p120 was detected with an anti-p120 monoclonal antibody (MAbp120) in most human malignant tumors (Freeman et al., Cancer Research, 48, 1244-1251, 1988). Stable transfection of the sense p120 cDNA caused malignant transformation of NIH/3T3 cells in vitro, and the antisense p120 constructs markedly delayed the growth of these transformed cells (Perlaky et al., Cancer Research, 52, 428-436, 1992). Several p120 antisense phosphorothioate oligonucleotides designed to hybridize with different regions of the p120 sequence were screened on human tumor cell lines in vitro. Marked growth inhibition of HeLa, LOX and HRCC cell lines was found, particularly with antisense p120 oligonucleotide ISIS 3466 in combination with N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); oligonucleotide ISIS 3466 is complementary to a non-translated region at the 3' end of the molecule. Preliminary in vivo studies on human LOX ascites tumor in nude mice showed marked inhibitory effects on tumor growth by the antisense oligonucleotide ISIS 3466 in the presence of DOTMA when treated on alternate days.

Cellular pharmacology of p120 antisense oligodeoxynucleotide phosphorothioate ISIS 3466.

Previous studies demonstrated that an antisense phosphorothioate oligodeoxynucleotide, ISIS 3466, to the human nucleolar p120 protein, markedly inhibited the growth of human tumor cell lines in vitro and inhibited the growth of the human LOX tumor in vivo in an i.p./i.p. model, in the presence of DOTMA (Perlaky et al., Anti-Cancer Drug Design 8:3-14, 1993). In vitro, DOTMA enhanced the effect of the antisense oligodeoxynucleotide was associated with the LOX cells after 4 hr treatment than in the absence of DOTMA. A 100-fold higher concentration of the oligodeoxynucleotide was required to introduce the same amount of oligodeoxynucleotide into the cells in the absence of DOTMA than in the presence of DOTMA. Kinetic analysis showed that the cell-associated oligodeoxynucleotide accumulated rapidly and reached a plateau after 1 hr incubation. When these cells were placed in a complete medium without the oligodeoxynucleotide, there was a 50% decrease in the oligodeoxynucleotide after 21 hr. A 35% reduction of p120 mRNA and a 50% reduction of p120 protein was found after ISIS 3466 treatment. Further study is needed to explore the tumor-inhibitory mechanisms of the effects of antisense oligodeoxynucleotide ISIS 3466.

A region of antisense RNA from human p120 cDNA with high homology to mouse p120 cDNA inhibits NIH 3T3 proliferation.

The human nucleolar p120 protein is a proliferation-associated antigen which is expressed in G1 and peaks during the early S phase of the cell cycle. Overexpression of the human p120 protein caused the transformation of NIH 3T3 cells and expression of an antisense p120 construct inhibited the growth of NIH 3T3 cells (Perlaky et al., Cancer Res., 52:428-436, 1992). The middle region of the antisense p120 RNA was found to be almost as inhibitory as the full length antisense construct but the 5' and 3' antisense portions did not affect NIH 3T3 cell proliferation. After the mouse p120 complementary DNA was cloned and sequenced, comparison with the human p120 complementary DNA showed a striking conservation of 85% of the nucleotide sequence and 96% of the amino acid sequence. The two ends of the p120 molecule had less homology in their nucleotide and amino acid sequences. Based on this homology, the observed inhibitory effects of the middle portion of antisense human p120 RNA may be related to suppression of mouse p120 expression by RNA:RNA duplex formation. The high evolutionary conservation of the middle region suggests it has a critical role for the function of this protein.

Increased growth of NIH/3T3 cells by transfection with human p120 complementary DNA and inhibition by a p120 antisense construct.

The human nucleolar antigen p120 was detected with an anti-p120 monoclonal antibody in most human malignant tumors but not in most resting human tissues (J. W. Freeman et al., Cancer Res., 48: 1244-1251, 1988) and has been used as a prognostic tumor marker in breast cancer patients (J. W. Freeman et al., Cancer Res., 51: 1973-1978, 1991). After the complementary DNA and gene for the human p120 protein were isolated and sequenced (review: H. Busch, Cancer Res., 50: 4830-4838, 1990), constructs were prepared to study the expression of the sense p120 and its antisense, p021 message. NIH/3T3 cells were transfected by electroporation with pSVX plasmids containing either the p120 complementary DNA (pSVX120) or the antisense, p021 DNA (pSVX021), and clones containing these constructs were selected. The expression of p120 or p021 in these constructs was regulated by Moloney murine leukemia virus long terminal repeats. In pSVX120-transfected NIH/3T3 cells, the expressed human p120 protein was localized to the nucleoli as shown by anti-p120 monoclonal antibody immunofluorescence. Expression of the p120 message and protein was confirmed by Northern (mRNA) and Western (protein) blots. Transfection of the p120 complementary DNA in sense orientation caused malignant transformation of NIH/3T3 cells in vitro and produced rapidly growing tumors in nude mice. Transfection of the antisense p120 constructs markedly delayed the growth of these tumors in vitro and in vivo (L. Perlaky et al., Proc. Am. Assoc. Cancer Res., 32: 1682, 1991). When transformed 3T3/pSVX120 cells were transfected with an inducible antisense p120 construct (pMSG021), dexamethasone induction decreased the growth rate by 62%, and the cell line returned to its normal phenotype. Northern blot analysis showed a decreased level of p120 mRNA, and the immunofluorescence was also markedly reduced.

Heat-induced morphological alterations in non-tolerant and thermotolerant cells.

CHO cells were heated at 43 degrees C or 45 degrees C for various durations up to 300 min. Survival values varied from 5 x 10(-1) to 10(-7). Unheated, non-tolerant control cells were compared with cells made thermotolerant (TT) by incubating at 37 degrees C for 6 or 12 h after treatment with either sodium arsenite (100 microM-As) or 45.5 degrees C for 10 min, respectively. Groups also were included in which heat-induced TT cells were heated at 43 degrees C for 5 h immediately before they were challenged at 45 degrees C; in these groups, cycloheximide was sometimes added to inhibit protein synthesis before and/or during heating at 43 degrees C. Morphological alterations were quantified immediately and at various times after heating by using phase-contrast microscopy to determine the percentage of cells that were severely blebbed and rounded. About 800 cells were analysed per datum point. When effects of heat on thermotolerant cells were compared with effects of heat on non-tolerant cells, heat-induced thermotolerance (HTT) was observed by an increase in survival, and by a reduction in the percentage of cells with morphological alteration observed immediately after the challenging heat. After the As treatment, very little thermotolerance was observed for morphological alterations immediately after the challenging heat, although thermotolerance was observed for survival. However, as the cells were incubated for 12 or 24 h at 37 degrees C after the challenging heat treatment, recovery from morphological alterations was observed in the As-TT cells. Possible mechanisms for the difference between HTT and As-TT are discussed.