A point mutation in the c-myc locus of a Burkitt lymphoma abolishes binding of a nuclear protein.

A 20-base pair region in the first intron of the human c-myc gene was identified as the binding site of a nuclear protein. This binding site is mutated in five out of seven Burkitt lymphomas sequenced to date. To investigate the protein-recognition region in greater detail, the abnormal c-myc allele from a Burkitt lymphoma line (PA682) that carries a t(8;22) chromosomal translocation was used. A point mutation in the binding region of the PA682 c-myc DNA abolished binding of this nuclear protein. This protein may be an important factor for control of c-myc expression, and mutations in its recognition sequence may be associated with c-myc activation in many cases of Burkitt lymphoma.

Transfection of v-rasH DNA into MCF-7 human breast cancer cells bypasses dependence on estrogen for tumorigenicity.

The natural history of estrogen-responsive breast cancers often involves a phenotypic change to an estrogen-unresponsive, more aggressive tumor. The human breast cancer cell line, MCF-7, which requires estradiol for tumor formation in vivo and shows growth stimulation in response to estradiol in vitro, is a model for hormone-responsive tumors. The v-rasH onc gene was transfected into MCF-7 cells. The cloned MCF-7ras transfectants, which expressed the v-rasH messenger RNA and v-rasH p21 protein (21,000 daltons), were characterized. In contrast to the parental cell line, MCF-7ras cells no longer responded to exogenous estrogen in culture and their growth was minimally inhibited by exogenous antiestrogens. When tested in the nude mouse, the MCF-7ras cells were fully tumorigenic in the absence of estrogen supplementation. Thus, cells acquiring an activated onc gene can bypass the hormonal regulatory signals that trigger the neoplastic growth of a human breast cancer cell line.

Three distinct genes in human DNA related to the transforming genes of mammalian sarcoma retroviruses.

Southern blot hybridization was used to identify human and other vertebrate DNA sequences that were homologous to cloned DNA fragments containing the oncogenic nucleic acid sequences of three different type C mammalian retroviruses (simian sarcoma virus, the Snyder-Theilen strain of feline sarcoma virus, and the Harvey strain of murine sarcoma virus). Each onc gene counterpart has a single genetic locus, which probably contains non-onc intervening sequences. The human DNA sequences may represent genes important to cell growth or cell differentiation, or both. Their identification and isolation may allow elucidation of their role in these processes and in neoplasias.

Proviral DNA of a retrovirus, human T-cell leukemia virus, in two patients with AIDS.

The acquired immune deficiency syndrome (AIDS) is characterized by T-lymphocyte dysfunction and is frequently accompanied by opportunistic infections and Kaposi's sarcoma. Human T-cell leukemia virus (HTLV) is associated with T-cell malignancies and can transform T lymphocytes in vitro. In an attempt to find evidence of HTLV infection in patients with AIDS, DNA from samples of peripheral blood lymphocytes from 33 AIDS patients was analyzed by Southern blot-hybridization with a radiolabeled cloned HTLV DNA probe. Analysis of DNA from both the fresh (uncultured) lymphocytes and from T cells cultured with T-cell growth factor revealed the presence of integrated HTLV proviral sequences in lymphocytes from two of the patients, both of whom had antibody to HTLV. The proviral sequences could not be detected in blood samples obtained from these individuals at a later date, consistent with the possibility that the population of infected cells had become depleted.

5' viral and human cellular sequences corresponding to the transforming gene of simian sarcoma virus.

The 5' nucleotide sequences of the transforming gene of simian sarcoma virus (v-sis) and its human cellular homolog (c-sis) were compared. A short homology was found between helper virus and cellular DNA sequences at the junction of v-sis and c-sis, which may have had a role in the original recombination event leading to the generation of simian sarcoma virus.

Isolation of human T-cell leukemia virus in acquired immune deficiency syndrome (AIDS).

Several isolates of a human type-C retrovirus belonging to one group, known as human T-cell leukemia virus (HTLV), have previously been obtained from patients with adult T-cell leukemia or lymphoma. The T-cell tropism of HTLV and its prevalence in the Caribbean basin prompted a search for it in patients with the epidemic T-cell immune deficiency disorder known as AIDS. Peripheral blood lymphocytes from one patient in the United States and two in France were cultured with T-cell growth factor (TCGF) an shown to express HTLV antigens. Virus from the U.S. patient was isolated and characterized and shown to be related to HTLV subgroup I. The virus was also transmitted into normal human T cells from umbilical cord blood of a newborn. Whether or not HTLV-I or other retroviruses of this family with T-cell tropism cause AIDS, it is possible that patients from whom the virus can be isolated can also transmit it to others. If the target cell of AIDS is the mature T cell as suspected, the methods used in these studies may prove useful for the long-term growth of these cells and for the identification of antigens specific for the etiological agent of AIDS.

Sera from patients with the acquired immunodeficiency syndrome inhibit production of interleukin-2 by normal lymphocytes.

We studied the effects of sera from patients with the acquired immunodeficiency syndrome (AIDS) on interleukin-2 (IL-2) production to help elucidate the mechanism of immunodeficiency. Compared with sera from healthy controls, sera from AIDS patients suppressed phytohemagglutinin (PHA)-induced IL-2 production by normal blood mononuclear cells. Sera from homosexual contacts of AIDS patients and from adults with acute cytomegalovirus infection generally lacked this suppressive activity. The effect of the AIDS sera could not be attributed to absence of a stimulatory or nutritive factor, to inactivation of IL-2, to inhibition of the IL-2 assay, nor to increased turnover of IL-2. The suppressive effect of the sera was not mediated by radiosensitive or T8 antigen-bearing suppressor cells or by increased prostaglandin production or decreased interleukin-1 production. The sera acted directly on the groups of cells that produce IL-2, T cells and large granular lymphocytes; suppression occurred at an early, probably pretranslational, stage. When cells were incubated with AIDS sera and then washed, the suppressive effect persisted. The sera did not cause direct or complement-mediated cytotoxic effects on normal mononuclear cells nor did they suppress PHA-induced interferon production, nor proliferation of T lymphoblasts or lymphocyte lines. The suppressive effect was not mediated by interferon, cortisol, immunoglobulin G or M, or immune complexes. The activity was stable at pH 3, pH 10, and 60 degrees C; inactivated at 100 degrees C; and not ether extractable. Because IL-2 plays a central role in the development of many immune responses, the serum factor(s) that inhibits IL-2 production could contribute significantly to the immunodeficiency of AIDS.

Human gene (c-fes) related to the onc sequences of Snyder-Theilen feline sarcoma virus.

The onc gene (v-fes) of the acutely transforming feline sarcoma virus (Snyder-Theilen strain) has homologous cellular sequences (c-fes) in all vertebrate species, including humans. We isolated from a human DNA library recombinant phages containing overlapping c-fes sequences. The human c-fes locus spans a region of 3.4 kilobases and contains 1.4 kilobases of DNA homologous to the viral onc sequence interspersed with three intervening sequences.

DNA-binding sequence of the human prostate-specific homeodomain protein NKX3.1.

NKX3.1 is a member of the NK class of homeodomain proteins and is most closely related to Drosophila NK-3. NKX3.1 has predominantly prostate-specific expression in the adult human. Previous studies suggested that NKX3.1 exerts a growth-suppressive effect on prostatic epithelial cells and controls differentiated glandular functions. Using a binding site selection assay with recombinant NKX3.1 protein we identified a TAAGTA consensus binding sequence that has not been reported for any other NK class homeoprotein. By electromobility shift assay we demonstrated that NKX3.1 preferentially binds the TAAGTA sequence rather than the binding site for Nkx2.1 (CAAGTG) or Msx1 (TAATTG). Using mutated binding sites in competitive gel shift assays, we analyzed the nucleotides in the TAAGTA consensus sequence that are important for NKX3.1 binding. The consensus binding site of a naturally occurring polymorphic NKX3.1 protein with arginine replaced by cysteine at position 52 was identical to the wild-type binding sequence. The binding affinities of wild-type and polymorphic NKX3.1 for the TAAGTA consensus site were very similar, with values of 20 and 22 nM, respectively. Wild-type and polymorphic NKX3.1 specifically repressed transcription of luciferase from a reporter vector with three copies of the NKX3.1-binding site upstream from a thymidine kinase promoter. The data show that among NK family proteins NKX3.1 binds a novel DNA sequence and can behave as an in vitro transcriptional repressor.

Cytogenetic studies in human T-cell lymphoma virus (HTLV)-positive leukemia-lymphoma in the United States.

Cytogenetic studies were conducted on fresh and cultured cells from 11 patients with human T-cell leukemia virus-associated adult T-cell leukemia-lymphoma. Clones with abnormal karyotypes were detected in 9 of the 11 patients. Chromosome numbers were near-diploid in cells from all but 1 patient who also had a tetraploid clone. The chromosome abnormalities in these cells were extensive; numerous complex structural changes were seen in every chromosome pair. Structural abnormalities occurred most frequently in chromosome 6. The 6 patients with chromosome 6 deletions had breakpoints at bands q11, q13, q16q23, q21q23, q22q24, and q23q24. The characteristic clinical features of these 6 patients were aggressive course, short survival, poor response to chemotherapy, high white blood cell counts, hypercalcemia, and bone lesions, whereas cytogenetically abnormal patients without chromosome 6q deletions tended to have a more indolent course. The precise role of the 6q deletion cannot be established with certainty from these data. However, this abnormality appears to occur with a greater than expected frequency in this large cell aggressive lymphoma, in association with hypercalcemia and lytic bone lesions.

p53 abnormalities in primary prostate cancer: single-strand conformation polymorphism analysis of complementary DNA in comparison with genomic DNA. The Cooperative Prostate Network.

BACKGROUND: The reported frequency of mutation of the p53 tumor suppressor gene (also known as TP53) in human carcinomas of the prostate has varied widely, ranging from 3% to 42%. This variability may be a consequence of tumor heterogeneity and/or the use of different methods of analysis. Since p53 mutation has been associated with clinical outcome for a number of cancer types, determination of its true frequency in primary carcinomas of the prostate is important. PURPOSE: The principal aims of this study were as follows: 1) to validate the utility of detecting p53 gene mutations by means of polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis of complementary DNA (cDNA) (synthesized from prostate tissue RNA and 2) to study the concordance of RNA- and DNA-based PCR-SSCP assays in detecting p53 mutations in individual tumor fragments. METHODS: RNA and genomic DNA were isolated by means of standard techniques from specimens of 19 carcinomas of the prostate, selected on the basis of p53 data obtained in a previous analysis of cDNA (indicating that 14 were mutant and five were wild-type). RNA was converted into cDNA by means of reverse transcription (RT); the cDNA was then amplified by means of nonisotopic (i.e., nonradioactive) PCR, and the PCR products were subjected to SSCP analysis in polyacrylamide gels (RT-PCR-SSCP analysis). Genomic DNA was examined by means of SSCP analysis of isotopically labeled (32PO4) PCR products (DNA-PCR-SSCP analysis). In both approaches, the protein coding region of the p53 gene was divided into multiple, smaller fragments for study. PCR products exhibiting abnormal migration in SSCP gels were subjected to direct nucleotide sequencing or to cloning and sequencing of multiple clones. RESULTS: RT-PCR-SSCP and DNA-PCR-SSCP identified p53 gene abnormalities in 15 of the 19 selected carcinomas, including one previously reported to be wild-type for p53. Overall, PCR-SSCP analysis identified 18 p53 fragments with abnormalities; three carcinomas showed two abnormalities each. Six (33%) of the 18 abnormalities were detected by both RT-PCR-SSCP and DNA-PCR-SSCP, 10 (56%) were detected by RT-PCR-SSCP alone, and two (11%) were detected by DNA-PCR-SSCP alone. The 18 abnormalities were caused by 20 changes in the sequence of the p53 gene; in one carcinoma, double mutations in two individual p53 exons were identified. CONCLUSIONS AND IMPLICATIONS: PCR-SSCP analysis of both RNA and DNA allows the detection of more mutations than the analysis of either alone. Some primary carcinomas of the prostate contain more than one altered p53 gene, consistent with the possibility of intratumoral heterogeneity of mutation of this gene. For comprehensive analysis of p53 mutations in carcinomas of the prostate, and perhaps in other tumor tissues, SSCP analysis of cDNA should be used in combination with SSCP analysis of genomic DNA.

Effects of steroid hormones and peptide growth factors on protooncogene c-fos expression in human breast cancer cells.

To investigate if the estrogen control of the tumorigenic phenotype of breast cancer cells was mediated through activation of the c-fos protooncogene, we examined the expression of this oncogene in MCF-7 cells. In cells synchronized by double thymidine blockade, the peptide growth factors transforming growth factor alpha and epidermal growth factor increased c-fos mRNA levels 6-fold above controls after 30 min of treatment. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, increased c-fos mRNA levels 4- to 5-fold above control. 17 beta-Estradiol, a growth stimulator, increased c-fos mRNA levels less than 2-fold above control levels, while progesterone, vitamin D3, dihydrotestosterone, and dexamethasone had little effect on c-fos mRNA levels. In contrast, 17 beta-estradiol treatment initially diminished the c-myc RNA level after 30 min of treatment and resulted in an elevation of c-myc by 2.5 h after initiation of treatment. We conclude that c-fos induction in these cells is growth related and accompanies stimulation by transforming growth factor alpha and epidermal growth factor. 17 beta-Estradiol, on the other hand, induced much smaller increases in c-fos mRNA levels, suggesting an alternative or more complex mechanism of cellular stimulation.

Beta-catenin mutations in human prostate cancer.

Beta-catenin plays essential roles in both intercellular adhesion and signal transduction. As a signaling molecule, beta-catenin supplies an activating domain to the T-cell factor/lymphoid enhancer-binding factor family of DNA-binding proteins and activates gene transcription. Posttranslational stabilization of beta-catenin, leading to elevated protein levels and constitutive gene activation, has been proposed as an important step in oncogenesis. Stabilization of beta-catenin can occur through mutation to highly conserved amino acids encoded in exon 3 of the beta-catenin gene (CTNNB1). To determine whether this pathway of malignant transformation is important in prostate cancer, we analyzed 104 prostate cancer tissue specimens, 4 prostate cancer cell lines, and 3 prostate tumor xenografts for activating mutations in exon 3 of CTNNB1. Mutations were detected in 5 of the 104 prostate cancer tissue samples. Four of the five mutations involved serine or threonine residues implicated in the degradation of beta-catenin. A fifth tumor had a mutation at codon 32, changing a highly conserved aspartic acid to a tyrosine. Mutational analysis of multiple regions from several tumor samples showed that the beta-catenin mutations were present focally and therefore may occur during tumor progression.

Radiation-induced apoptosis mediated by retinoblastoma protein.

The role of the retinoblastoma gene product, RB, in transmitting the signals of apoptosis is unclear, but RB is considered to be antiapoptotic because RB mediates cell cycle arrest that also can interrupt intracellular signaling pathways leading to apoptosis. Gamma-radiation can cause apoptosis, the process of programmed cell death, via several mechanisms including DNA damage, ceramide production, and the generation of free radical oxygen species. We investigated the effect of RB on radiation-induced apoptosis by restoring normal RB expression in DU-145 prostate cancer cells that have one deleted and one truncated RB gene. DU-145 cells are highly resistant to apoptosis induced either by radiation or by the addition of ceramide. Two independently derived RB-positive DU-145 derivative cell lines underwent apoptosis after irradiation or exposure to the cell permeable C2-ceramide. Apoptosis in the RB-positive cell lines was not associated with major changes in the cell cycle response to irradiation. RB-mediated apoptosis occurred in the absence of expression of caspases 8, 6, 3, and 7 and without detectable cleavage of poly(ADP)ribose polymerase. However, a specific inhibitor of serine proteases, Na-p-Tosyl-L-lysyl-chloromethyl ketone, inhibited radiation-induced apoptosis in DU-145 cells expressing RB. Radiation-induced apoptosis was preceded by an increase in JUN protein expression and accompanied by activation of the stress-related JUN kinase. Our data show that RB is proapoptotic in DU-145 cells and acts upstream of JUN expression and JNK activation.

Beta-catenin affects androgen receptor transcriptional activity and ligand specificity.

beta-Catenin is a multifunctional molecule with important roles in intercellular adhesion and signal transduction. We reported previously that beta-catenin is mutated in human prostate cancer. In this study, we investigated the role of beta-catenin mutations on androgen receptor (AR) signaling. beta-Catenin significantly enhanced androgen-stimulated transcriptional activation by the AR. beta-Catenin also increased AR transcriptional activation by androstenedione and estradiol and diminished the antagonism of bicalutamide. Coimmunoprecipitation of beta-catenin with AR from LNCaP prostate cancer cells showed that the two molecules are present in the same complex. The amount of beta-catenin in complex with AR was increased by androgen. These findings implicate beta-catenin in the regulation of AR function and support a role for beta-catenin mutations in the pathogenesis of prostate cancer.

Androgen blocks apoptosis of hormone-dependent prostate cancer cells.

Androgen plays a critical role in the promotion and growth of prostate cancer. Androgen ablation has an expanding role in prostate cancer treatment and is now used to improve the efficacy of radiation therapy in addition to its role in treatment of metastatic disease. Here we show that androgen interferes with induction of prostate cancer cell death induced by a variety of stimuli. The effect of androgen on cell death occurs predominantly by interference with caspase activation and the inhibition of caspase cleavage in both the extrinsic and intrinsic cell death pathways. Androgen inhibited apoptosis induced by both tumor necrosis factor alpha (TNF-alpha) and by Fas activation with or without concomitant irradiation. An antiapoptotic effect was seen in the presence of R1881, dihydrotestosterone, and also 17beta-estradiol within 24 h of death induction. Sustained inhibition of apoptosis at 72 h was seen only with R1881, dihydrotestosterone, cyproterone acetate, and hydroxyflutamide. Androgen treatment inhibited activation of caspases-8, -7, and -9 by TNF-alpha +/- irradiation. Androgen attenuated BAX expression and blocked appearance of the proapoptotic p18 fragment of BAX. Androgen also abrogated BID cleavage induced by TNF-alpha + irradiation that contributed to a decrease in cytochrome c egress from mitochondria induced by TNF-alpha +/- irradiation. There was also decreased mitochondrial depolarization in response to TNF-alpha + irradiation. Production of the proapoptotic lipid metabolite ceramide was not affected by androgen, but androgen acted downstream from ceramide generation because R1881 blocked cell-death induction by bacterial sphingomyelinase. Inhibition of phosphoinositol-3-kinase activity by wortmannin induced apoptosis that was also blocked by androgen, but there was no effect on protein levels or phosphorylation of AKT, indicating that R1881 did not interact with survival signaling of phosphoinositol-3-kinase. Lastly, androgen inhibited activation of nuclear factor-kappaB during death induction, but the effect of androgen on cell death was not mediated by interference with the nuclear factor-kappaB pathway. The data suggest that androgen induced blockade of caspase activation in both intrinsic and extrinsic cell death pathways and thereby was able to protect prostate cancer cells from apoptosis induced by diverse stimuli.

Tumor necrosis factor-alpha sensitizes prostate cancer cells to gamma-irradiation-induced apoptosis.

LNCaP prostate cancer cells are highly resistant to induction of programmed cell death by y-irradiation and somewhat sensitive to the death-inducing effects of tumor necrosis factor (TNF)-alpha. Simultaneous exposure of LNCaP cells to TNF-alpha and 8 Gy of irradiation was synergistic and resulted in a 3-fold increase of apoptotic cells within 72 h compared to TNF-alpha alone. It appeared that TNF-alpha sensitized the cells to irradiation because, when cells were irradiated 24 h after exposure to TNF-alpha, increased cell death was observed. In contrast, irradiation delivered 24 h prior to TNF-alpha exposure did not result in more cell death than after TNF-alpha alone. TNF-alpha induced expression of its own mRNA, but TNF-alpha mRNA induction was neither induced nor enhanced by irradiation. Activation of the transcription factor nuclear factor kappaB can be induced by TNF-alpha and has a modulating antiapoptotic effect. But enhancement of TNF-alpha-induced cell death by irradiation did not result from altered activation of nuclear factor kappaB. TNF-alpha treatment of LNCaP cells resulted in partial activation of caspase-8 and -6 but not caspase-3. There was only minimal poly(ADP-ribose) polymerase cleavage seen in LNCaP cells after exposure to both TNF-alpha and irradiation at 72 h, a time when 60% of the cells were apoptotic. Experiments with peptide inhibitors of cysteine and serine proteases suggested that caspases were the predominant mediators of apoptosis induced by TNF-alpha alone but that serine proteases contributed significantly to cell death induced by TNF-alpha plus irradiation. TNF-alpha increased production of ceramide in LNCaP cells 48 h after exposure. Although irradiation alone had no effect on ceramide production in LNCaP cells, TNF-alpha plus irradiation induced significantly more ceramide than TNF-alpha alone. Ceramide production did not occur immediately after exposure to TNF-alpha, but rather was delayed such that ceramide levels were increased only 24 h after exposure to apoptotic stimuli. Moreover, non-toxic levels of exogenous C2-ceramide sensitized LNCaP cells to irradiation similarly to TNF-alpha, suggesting that one mechanism by which LNCaP cells were sensitized to irradiation was by increased intracellular ceramide. Hence, ceramide generation is a critical component in radiation-induced apoptosis in human prostate cancer cells. Inhibition of ceramide generation may provide a selective advantage in the development of radioresistance in prostate cancer.

Growth properties and tumorigenesis of MCF-7 cells transfected with isogenic mutants of rasH.

MCF-7 human breast cancer cells are estrogen dependent for maximal in vitro growth and for tumor formation in nude mice, thus providing a useful model system to study mammary tumorigenesis. A clone of MCF-7 cells transfected with the v-rasH oncogene has been shown to form tumors in the absence of estradiol [Kasid et al., 1985, Science (Wash. DC), 228:725-728]. To extend this observation to more clones of v-rasH-expressing MCF-7 cells and to examine the effects of rasH mutation, we transfected MCF-7 cells with a construct encoding the human c-rasH protooncogene protein product and with three isogenic constructs encoding proteins containing point mutations: arg-12, thr-59, and arg-12 + thr-59 (v-rasH). We isolated several cell lines which produced levels of c-rasH and v-rasH p21 at 30- to 50-fold the levels of controls. We also isolated several cell lines producing the various mutants p21s. All of the transfected cell lines were estrogen-responsive for cell growth. Transfected cells containing high levels of rasH p21 had correspondingly high levels of growth in an anchorage-independent growth assay. Tumorigenesis studies in nude mice, however, showed that some, but not all of the cell lines expressing v-rasH, formed tumors in the absence of estradiol. Tumor formation did not correlate with the level of rasH p21 expression in these cell lines. No tumor formation in the absence of estradiol was observed for cell lines expressing single-mutated or unmutated forms of rasH.

Therapeutic approaches to patients with AIDS.

The immune systems of patients with acquired immunodeficiency syndrome are characterized by a profound defect in the number and function of helper/inducer T-lymphocytes, particularly at the level of soluble antigen recognition. Due to this selective yet profound defect in the immune system, these patients are prone to recurrent severe opportunistic infections and Kaposi's sarcoma. While therapies exist for some of these complications of acquired immunodeficiency syndrome, no effective therapies exist for the underlying immune defect of this syndrome. Reviewed here are some of our recent attempts at immune reconstitution in acquired immunodeficiency syndrome, using either whole scale immune reconstitution through the use of lymphocyte transfers and bone marrow transplantation or enhancement of the remaining immune systems with the T-cell-derived lymphokines interleukin-2 or immune gamma-interferon. In addition, recent advances in the therapy of Pneumocystis carinii pneumonia and disseminated cytomegalovirus disease are discussed.

Heterogeneous expression of erbB-2 messenger RNA in human breast cancer.

Amplification and mRNA expression of the erbB-2 gene was analyzed in 61 samples of primary human breast carcinoma. In the 57 samples where RNA could be isolated four different expression level groups were identified. Comparison of hybridization signal with that for beta-actin revealed that erbB-2 mRNA could not be detected in 6 of 57 samples (11%), was detected at normal levels in 32 of 57 samples (56%), showed 4- to 8-fold overexpression in 8 of 57 samples (14%), and showed 16- to 128-fold overexpression in 11 of 57 samples (19%). Examination of the DNA of the same set of samples revealed 6 of 61 samples (10%) with distinct gene amplification and 6 of 61 samples (10%) with possible gene amplification. The highest levels of erbB-2 overexpression were associated with gene amplification. Samples with 4- to 16-fold overexpression of the erbB-2 mRNA occurred without evident gene abnormalities. There was no association of erbB-2 expression or gene amplification with clinical stage of breast carcinoma or axillary lymph node involvement. The clear amplification of the erbB-2 gene may be associated with a significantly shorter time to treatment failure.