Identification of the p53 family-responsive element in the promoter region of the tumor suppressor gene hypermethylated in cancer 1.

The tumor suppressor gene hypermethylated in cancer 1 (HIC1), located on human chromosome 17p13.3, is frequently silenced in cancer by epigenetic mechanisms. Hypermethylated in cancer 1 belongs to the bric à brac/poxviruses and zinc-finger family of transcription factors and acts by repressing target gene expression. It has been shown that enforced p53 expression leads to increased HIC1 mRNA, and recent data suggest that p53 and Hic1 cooperate in tumorigenesis. In order to elucidate the regulation of HIC1 expression, we have analysed the HIC1 promoter region for p53-dependent induction of gene expression. Using progressively truncated luciferase reporter gene constructs, we have identified a p53-responsive element (PRE) 500 bp upstream of the TATA-box containing promoter P0 of HIC1, which is sequence specifically bound by p53 in vitro as assessed by electrophoretic mobility shift assays. We demonstrate that this HIC1 p53-responsive element (HIC1.PRE) is necessary and sufficient to mediate induction of transcription by p53. This result is supported by the observation that abolishing endogenous wild-type p53 function prevents HIC1 mRNA induction in response to UV-induced DNA damage. Other members of the p53 family, notably TAp73beta and DeltaNp63alpha, can also act through this HIC1.PRE to induce transcription of HIC1, and finally, hypermethylation of the HIC1 promoter attenuates inducibility by p53.

Retinoids. Structure-function relationship in normal and leukemic hematopoiesis in vitro.

Retinoids were studied both to identify what skeletal components are important in the modulation of normal and leukemic human myeloid clonal proliferation and differentiation in vitro and to elucidate the mechanism by which retinoids modulate proliferation of hematopoietic cells. Retinoids with a derivatized terminal carboxyl group were significantly less active than all-trans-retinoic acid, and those with the addition of two methyl groups to the cyclohexenyl ring of retinoic acid or substitution of its beta-cyclogeranylidene group with a 1,1,3,3-5-indanyl ring system were markedly more active than all-trans-retinoic acid. Five of the retinoids strongly inhibited clonal growth of the HL-60 and KG-1 human leukemic cell lines (50% inhibition in the range of 3 X 10(-10)-1 X 10(-8) M) and markedly stimulated normal human myeloid colony formation (granulocyte-macrophage colony-forming cells [GM-CFC] 150% stimulation in the range of 3 X 10(-9)-3 X 10(-8) M). Further studies suggested that: Common structural requirements of the retinoids were important in the modulation of both normal and leukemic hematopoiesis. The retinoids were able to inhibit leukemic proliferation without induction of differentiation of the neoplastic cells. Studies on normal human GM-CFC suggested that the retinoids did not act by themselves as a colony-stimulating factor (CSF), or by stimulating accessory cells to produce CSF, but either required earlier progenitor cells to become GM-CFC or enhanced the sensitivity of GM-CFC to the action of CSF.

1,25-Dihydroxyvitamin D3 modulates the expression of a lymphokine (granulocyte-macrophage colony-stimulating factor) posttranscriptionally.

We recently showed that 1,25(OH)2D3 sensitively inhibited the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in normal human mitogen-activated peripheral blood lymphocytes and in the human T lymphotropic virus I immortalized T cell line known as S-LB1 at the levels of both mRNA and protein. Using S-LB1 cells as a model system the present paper identifies at least in part the mechanisms by which 1,25(OH)2D3 regulates the expression of GM-CSF. Time-course studies demonstrated that by 6 and 48 h of exposure of S-LB1 cells to 1,25(OH)2D3 (10(-8) M) the GM-CSF mRNA levels were reduced by 50 and 90%, respectively. Studies using cycloheximide as a protein synthesis inhibitor showed that the inhibitory action of 1,25(OH)2D3 on GM-CSF expression was dependent on new protein synthesis. In vitro nuclear run-on assays demonstrated that 1,25(OH)2D3 (10(-8) M) did not change the rate of transcription of the GM-CSF gene. The t1/2 of GM-CSF mRNA, however, was profoundly reduced by 1,25(OH)2D3 when transcription was blocked by actinomycin D compared with the half-life of GM-CSF in the presence of actinomycin D alone (t1/2, less than 0.5 and 4 h, respectively). Taken together, these results demonstrate that 1,25(OH)2D3 regulates expression of the lymphokine GM-CSF posttranscriptionally by influencing the stability of GM-CSF mRNA.

Granulocyte-macrophage colony-stimulating factor. Sensitive and receptor-mediated regulation by 1,25-dihydroxyvitamin D3 in normal human peripheral blood lymphocytes.

We show that 1,25-dihydroxyvitamin D3 (1,25[OH]2D3), the most hormonally active metabolite of vitamin D3, modulates sensitively and specifically both the protein and messenger RNA accumulation of the multilineage growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF). The regulation of GM-CSF expression is seen in both normal human mitogen-activated T lymphocytes and T lymphocytes from a line (S-LB1) transformed with human T cell lymphotropic virus 1 (HTLV-1). In contrast, cells from a HTLV-1 transformed T lymphocyte line (Ab-VDR) established from a patient with vitamin D-resistant rickets type II with undetectable 1,25(OH)2D3 cellular receptors are resistant to the action of 1,25(OH)2D3. Inhibition of GM-CSF expression by 1,25(OH)2D3 can occur independently of interleukin 2 regulation and is probably mediated through cellular 1,25(OH)2D3 receptors. We conclude that 1,25(OH)2D3 may be important in the physiology of hematopoiesis.

Posttranscriptional stabilization underlies p53-independent induction of p21WAF1/CIP1/SDI1 in differentiating human leukemic cells.

p21WAF/CIP1/SDI1 is a recently identified gene expressed in cells harboring wild-type but not mutant p53 gene. It encodes a nuclear protein of 21 kD which inhibits cyclin-dependent kinase activity. Constitutive p21WAF1/CIP1/SDI1 mRNA expression was detected in neoplastic cells from patients with various hematological malignancies as well as in normal bone marrow mononuclear cells and in myeloid and lymphoid cell lines independent of their p53 status. Induced differentiation of the p53-deficient promyelocytic HL-60 cells along the monocytic lineage by phorbol ester or 1a,25 dihydroxyvitamin D3 resulted in a marked increase of both p21WAF1/CIP1/SDI1 mRNA and protein expression due to enhanced mRNA stability. Differentiation towards the granulocytic lineage by all-trans retinoic acid or dimethylsulfoxide failed to produce this effect. p21WAF1/CIP1/SDI1 is an immediate early gene since its upregulation occurred independently of de novo protein synthesis. The induction of p21WAF1/CIP1/SDI1 expression and its regulation in p53-deficient differentiating leukemic cells support the idea of an additional, p53-independent role of p21WAF1/CIP1/SDI1 in human hematopoiesis.

Clonal analysis of human tumors with M27 beta, a highly informative polymorphic X chromosomal probe.

The clonality of human tumors can be studied by X inactivation/methylation analysis in female patients heterozygous for X-linked DNA polymorphisms. We present a detailed study on clonal tumor analysis with M27 beta, a highly informative probe detecting a polymorphic X chromosomal locus, DXS255. The polymorphism detected at this locus is due to variable numbers of tandem repeats. The rate of constitutional heterozygosity detected by M27 beta was 88%. Normal tissue from gastrointestinal mucosa and thyroid showed random, hence polyclonal, patterns. Nonrandom clonal X inactivation was detected in all 22 malignant neoplasms that had been shown to be clonal by other DNA markers, such as antigen receptor gene rearrangements or clonal loss of heterozygosity at 17p and other loci. 16/48 normal blood leukocyte samples (33%) showed considerably skewed X inactivation patterns. Comparison of blood leukocytes and normal tissue indicated that in a given individual, X inactivation patterns may be tissue specific. M27 beta was used to study the clonal composition of 13 benign thyroid nodules from 12 multinodular goiters with rapid recent growth, traditionally termed "adenomas." Nine of them were clonal, whereas four nodules and tissue from a case of Graves' goiter were not, indicating that some, but not all, such thyroid nodules may represent true clonal neoplasms. The M27 beta probe permits one to study the clonal composition by the X inactivation approach of a wide variety of solid tumors from most female patients. As a control, normal tissue homologous to the tumor type of interest is preferable to DNA from blood leukocytes, since the latter may show nonrandom X inactivation patterns in a fairly high proportion of cases. M27 beta may, therefore, be of limited use for the clonal analysis of neoplasms derived from hematopoietic cells.

Transcriptional and posttranscriptional modulation of myeloid colony-stimulating factor expression by tumor necrosis factor and other agents.

Granulocyte (G) and granulocyte-macrophage (GM) colony-stimulating factors (CSF) are necessary for proliferation and differentiation of myeloid hematopoietic cells. Fibroblasts stimulated by tumor necrosis factor alpha (TNF alpha) and several other agents are a rich source of these CSF. The GM-CSF synthesized by these cells had the same molecular weight and glycosylation pattern as that produced by activated T lymphocytes, as shown by [35S]methionine labeling studies. Northern (RNA) blot analysis showed that the fibroblasts had trace levels of G- and GM-CSF mRNA. Both G- and GM-CSF mRNA concentrations coordinately increased after exposure of the cells to TNF alpha (greater than or equal to 5 ng/ml), 12-O-tetradecanoylphorbol 13-acetate (TPA) (greater than or equal to 5 x 10(-10) M), or cycloheximide (20 micrograms/ml). Both TNF alpha and TPA increased levels of G- and GM-CSF mRNA in the absence of new protein synthesis. Transcriptional run-on studies demonstrated that fibroblasts constitutively transcribed GM-CSF, and transcription was enhanced 3.0-fold by TNF alpha and 2.5-fold by TPA and was unchanged by cycloheximide. The stability of G- and GM-CSF transcripts was determined after exposure of the cells to actinomycin D; the half-lives of G- and GM-CSF mRNA in unstimulated cells were less than 0.25 h and were increased 2- to 16-fold in cells cultured with TNF, TPA, or cycloheximide. In summary, both transcriptional and posttranscriptional signals acted coordinately to modulate the levels of G- and GM-CSF mRNAs in fibroblasts.

Cytogenetic response to prior treatment with interferon-alpha is predictive for survival after allogeneic hematopoietic stem cell transplantation in chronic myeloid leukemia.

We investigated the impact of a cytogenetic response (CyR) to IFN prior to and at the time of allogeneic hematopoietic stem cell transplantation (HSCT) on transplant-related mortality (TRM), relapse rate and survival probability after HSCT in 162 transplanted patients with chronic myeloid leukemia. One-hundred-one patients (62.3%) achieved a CyR prior to HSCT. Survival probabilities were higher in patients, who achieved any CyR prior to HSCT than in patients without CyR (63.6 vs 49.2%: P = 0.019). Survival probabilities in patients, who achieved a major CyR were better than in patients with minimal and minor CyR or in patients with no CyR (69.4 vs 58.8% vs 49.2%: P = 0.040). TRM and survival of chronic phase patients without CyR at the time of HSCT were similar to that of patients transplanted in advanced phase. Both groups combined had an outcome inferior to patients with at least minimal CyR (TRM, Gray test: P = 0.016, survival, log-rank test: P = 0.002). Univariate and multivariate analyses identified CyR prior to or at HSCT as a strong and independently favorable prognostic factor. We therefore conclude that allogeneic HSCT in CyR should be investigated prospectively as an alternative treatment option in defined patient groups.

Human small cell lung cancer expresses the octamer DNA-binding and nervous system-specific transcription factor N-Oct 3 (brain-2).

Small cell lung cancer (SCLC) cells express several characteristics of neuronal cells, including synthesis of neuropeptides and expression of the respective receptors. Establishment and maintenance of the neuronal phenotype of SCLC may depend on expression of gene transcription factors inherent to the central nervous system. The present study shows the nervous system-specific transcription factor N-Oct 3 (brain-2) to be expressed in all 13 SCLC cell lines investigated. Furthermore, N-Oct 3 (brain-2) was also found in SCLC-derived skin metastasis. In contrast, in extracts and RNA of non-SCLC cell lines and non-SCLC tumor tissues, such as lung squamous, large cell, and adenocarcinoma, expression of N-Oct 3 (brain-2) was not detectable. These data support the concept that SCLC cells derive from the neuroectodermal cell lineage since expression of N-Oct 3 (brain-2) protein is highly abundant at the neural tube stage and in the adult restricted to the neuroectodermal cell lineage.

Somatic mutations detected by mini- and microsatellite DNA markers reveal clonal intratumor heterogeneity in gastrointestinal cancers.

We investigated clonal intratumor heterogeneity by comparing different areas of each tumor in 20 gastrointestinal cancers from female patients (1 esophageal cancer, 5 stomach cancers, and 14 colorectal cancers). In all 19 cases informative for X-inactivation analysis with the M27 beta and/or the phosphoglycerate kinase probes, the tumors were clonal. Separate areas from a given tumor showed identical X-inactivation patterns, providing evidence for its single-cell origin. Of 20 cancers, 11 showed p53 gene mutations (base pair insertions, point mutations, and one case of a base pair deletion) in exons 5-8. A particular p53 gene mutation was identical in all tumor areas investigated per case. The minisatellite probes detected loss of heterozygosity or new mutant alleles at 1p33, 1q21, 5q35, 17p13, or 18q21. In seven cases mutations at particular loci were restricted to one or two areas per tumor, while in another seven cases they were common to all tumor areas. Loss of heterozygosity or new alleles detected at the microsatellite loci D2S123, D3S1611, D5S107, D17S261, or D18S34 [(CA)n repeats] were common to all tumor areas in 7 of 19 cases. In another seven cases, however, microsatellite mutations at these loci were restricted to one to three areas per tumor. Tracing clonal intratumor heterogeneity would permit one to study the hierarchy of mutational events in cancers where no premalignant lesions can be harvested. Most important, our study indicates that clonal intratumor heterogeneity might lead to sampling errors in the molecular diagnosis of cancer biopsy specimens when using mini- or microsatellite markers.

Distinct expression patterns of the p53-homologue p73 in malignant and normal hematopoiesis assessed by a novel real-time reverse transcription-polymerase chain reaction assay and protein analysis.

The role of the recently identified first p53-homologue, p73, in neoplastic transformation is unknown. To elucidate p73 gene expression in hematopoiesis, we investigated samples from chronic myeloid leukemia (CML) and acute myeloid leukemia patients, leukemia cell lines, as well as mature and immature normal hematopoietic cells by real-time quantitative RT-PCR and Western blot analysis. We found a distinct p73 expression profile with highest p73 mRNA transcript levels in hematopoietic malignancies such as CML blast crisis and acute myelogenous leukemia versus CML chronic phase and normal controls. Mono- and biallelic p73 expression was found in both normal and malignant hematopoiesis. p73 protein was expressed at various levels in leukemia samples and cell lines but could not be detected in any normal controls tested. Our results point to a distinct yet undefined role of p73 in the pathogenesis of myeloid neoplasms.

Aberrant FHIT mRNA transcripts are present in malignant and normal haematopoiesis, but absence of FHIT protein is restricted to leukaemia.

Aberrant FHIT mRNA transcripts are present in malignant and normal haematopoiesis, but absence of FHIT protein is restricted to leukaemia Alterations of the recently cloned fragile histidine triad (FHIT) gene at chromosome 3p14.2 are frequent in a variety of solid tumours and cancer cell lines. Based on these findings, FHIT has been proposed as a putative tumour-suppressor gene. We evaluated the mRNA expression of the FHIT gene in samples from 55 patients with various haematological malignancies (21 AML, 8 CML, 10 CLL, seven low-grade and nine high-grade Non-Hodgkin's lymphomas), in a panel of 16 leukaemia cell lines, in normal mature haematopoietic cells of both myeloid and lymphoid lineage, as well as in CD34+ haematopoietic progenitor cells. Aberrant FHIT mRNA transcripts were observed in 14/16 (88%) leukaemia cell lines, 43/55 (78%) primary haematological neoplasms, but also in 17/22 (77%) normal controls. 1/16 (6%) cell lines and 7/55 (13%) neoplasms did not express any FHIT mRNA. cDNA sequencing revealed exonic deletions, small DNA insertions and combinations of both. Analysis of genomic DNA showed gene deletions in two myeloid leukaemia cell lines. In contrast to all normal types of haematopoietic cells, FHIT protein was clearly reduced or absent in 8/18 (44%) neoplastic samples tested. Our data indicate that whilst aberrant FHIT mRNA transcripts are seen both in normal and malignant cells, lack of FHIT protein is restricted to leukaemia. Absent FHIT protein expression might contribute to leukaemogenesis.

Expression of p16INK4a/p16alpha and p19ARF/p16beta is frequently altered in non-small cell lung cancer and correlates with p53 overexpression.

The CDKN2 locus expresses two different mRNA transcripts, designated alpha and beta. The protein product of the alpha transcript is the cell cycle inhibitor and tumour suppressor p16INK4a. The beta transcript is translated in an alternate reading frame (ARF) and in humans encodes a 15 kDa protein (p19ARF). Immunohistochemical and Western analysis of p16INK4a has shown that the protein is downregulated in a significant number of tumours, but less is known on the expression of the p19ARF. We have examined the expression of p16INK4a and p19ARF in resectable non-small cell lung cancer (NSCLC) by immunostaining (n=49) and multiplex RT-PCR (n=28). In order to investigate the mechanism responsible for p16INK4a downregulation, exon 1alpha methylation was analysed in a PCR-based assay. Of 49 tumours examined by immunostaining, 24 and 20 tumours expressed p16INK4a and p19ARF at nil to low levels, respectively. p19ARF was localized primarily to the nuclei of tumour cells, but was also seen to varying degrees in nuclei of lymphocytes, chondrocytes, fibroblasts, and epithelial cells. No tumour with normal p16INK4a had decreased p19ARF expression. Among 16 tumours with nil to low p16INK4a expression, 11 tumours exhibited full methylation of at least one site within exon 1alpha and these tumours showed normal p19ARF expression. In contrast, no methylation of exon 1alpha was observed in five tumours which also lacked p19ARF. In normal lung, p16INK4a and p19ARF were not expressed at detectable levels, the multiplex RT-PCR results were balanced, and sites within exon 1alpha were strongly methylated. In tumours, imbalanced multiplex RT-PCR data (p16INK4a

Human delta Np73 regulates a dominant negative feedback loop for TAp73 and p53.

Inactivation of the tumour suppressor p53 is the most common defect in cancer cells. p53 is a sequence specific transcription factor that is activated in response to various forms of genotoxic stress to induce cell cycle arrest and apoptosis. Induction of p53 is subjected to complex and strict control through several pathways, as it will often determine cellular fate. The p73 protein shares strong structural and functional similarities with p53 such as the potential to activate p53 responsive genes and the ability to induce apoptosis. In addition to alternative splicing at the carboxyl terminus which yields several p73 isoforms, a p73 variant lacking the N-terminal transactivation domain (Delta Np73) was described in mice. In this study, we report the cloning and characterisation of the human Delta Np73 isoforms, their regulation by p53 and their possible role in carcinogenesis. As in mice, human Delta Np73 lacks the transactivation domain and starts with an alternative exon (exon 3'). Its expression is driven by a second promoter located in a genomic region upstream of this exon, supporting the idea of two independently regulated proteins, derived from the same gene. As anticipated, Delta Np73 is capable of regulating TAp73 and p53 function since it is able to block their transactivation activity and their ability to induce apoptosis. Interestingly, expression of the Delta Np73 is strongly up-regulated by the TA isoforms and by p53, thus creating a feedback loop that tightly regulates the function of TAp73 and more importantly of p53. The regulation of Delta Np73 is exerted through a p53 responsive element located on the Delta N promoter. Expression of Delta Np73 not only regulates the function of p53 and TAp73 but also shuts off its own expression, once again finely regulating the whole system. Our data also suggest that increased expression of Delta Np73, functionally inactivating p53, could be involved in tumorogenesis. An extensive analysis of the expression pattern of Delta Np73 in primary tumours would clarify this issue.

Randomized study of recombinant human granulocyte colony-stimulating factor after high-dose chemotherapy and autologous bone marrow transplantation for high-risk lymphoid malignancies.

PURPOSE: The aim of this prospective randomized trial was to examine the efficacy and safety of filgrastim after high-dose chemotherapy and autologous bone marrow transplantation (ABMT). PATIENTS AND METHODS: Patients with poor-risk non-Hodgkin's lymphoma or relapsed Hodgkin's disease were treated in a randomized, open-label trial to study the use of filgrastim as an adjunct to high-dose chemotherapy and ABMT. Of 43 assessable patients, 19 were randomized to receive filgrastim by continuous subcutaneous infusion at a dose of 10 micrograms/kg/d, 10 to filgrastim 20 micrograms/kg/d, and 14 to a parallel control group that received no filgrastim after ABMT. RESULTS: For all filgrastim-treated patients analyzed together, the median time to neutrophil recovery > or = 0.5 x 10(9)/L after the day of ABMT was significantly accelerated to 10 days compared with 18 days in control patients (P = .0001). The median number of platelet transfusions was identical in both groups. Clinical parameters, including the median number of days with fever (1 v 4, P = .0418) and neutropenic fever (5 v 13.5, P = .0001) were significantly shorter in the filgrastim than in the control group. The number of days on intravenous antibiotics and duration of hospitalization were also shorter in the treated groups; however, the differences did not reach statistical significance. For patients treated with the two different dose levels of filgrastim, the neutrophil recovery and clinical results were similar. Filgrastim-associated toxicity appeared to be minimal, with five adverse events considered at least possibly related to filgrastim: two in the higher-dose group and three in the lower-dose group. All of these were rated moderate, except one case of severe bone pain that did not preclude continued filgrastim treatment at a lower dose. Survival and relapse-free survival were similar for control and filgrastim-treated patients. CONCLUSION: Taken together, the results of this first randomized study support the role of filgrastim given as an adjunct to ABMT in accelerating neutrophil recovery, as well as in reducing treatment-related morbidity and overall duration of the treatment procedure.

Cytosine demethylation of the proteinase-3/myeloblastin primary granule protease gene during phagocyte development.

Proteinase-3/Myeloblastin (Mbn) is a neutral serine protease and a major constituent of the primary granules of myeloid cells. It can degrade extracellular matrix proteins and has been discussed as a key factor for the initiation of terminal differentiation in promyelocytic cells. Regulation of Mbn closely parallels that of another major primary granule protein, myeloperoxidase (MPO). We examined the expression and DNA methylation of Mbn in a model of in vitro differentiation of CD34+ enriched peripheral blood progenitor cells (PBPCs), and in various other myeloid and non-myeloid tissues. Mbn mRNA was undetectable in uncultured PBPCs but was upregulated during their in vitro differentiation. Its expression was enhanced in the presence of G-CSF. Mbn expression was also detected in several myeloid cell lines but not in mature granulocytes, monocytes and macrophages. Partial demethylation at a CpG site within Mbn intron 1 (analyzed by restriction with SmaI) was observed during continued in vitro differentiation of PBPCs. This site was fully demethylated in mature granulocytes, monocytes and macrophages. Variable methylation of this site and a second SmaI site located upstream of the putative Mbn promoter region was present in other myeloid and non-myeloid tissues examined.

Expression and regulation of G1 cell-cycle inhibitors (p16INK4A, p15INK4B, p18INK4C, p19INK4D) in human acute myeloid leukemia and normal myeloid cells.

In hematological malignancies, structural alterations of genes for G1-specific cyclin-dependent kinases inhibitors (CKIs) have been extensively investigated. G1-CKIs might play an important role not only as tumor suppressor genes but also in cellular differentiation. We examined constitutive and differentiation-induced expression and regulation of the four members of the G1-CKI family p16INK4A, p15INK4B, p18INK4C and p19INK4D in acute myeloid leukemia as well as their expression in normal granulocytes and monocytes. p18INK4C and p19INK4D mRNA were expressed constitutively at high levels in seven myeloid cell lines and 16 AML patient samples, whereas expression of p15INK4B mRNA was very low and only detectable by nested RT-PCR analysis. During phorbol ester-induced monocytic differentiation of leukemic HL-60 cells expression of particular G1-CKIs was disparately regulated. This process was associated with growth arrest of the majority of the cells (> or = 80%) in G1/G0, and in parallel p15INK4B were upregulated whereas p18INK4C and p19INK4D expression was downregulated. In contrast, granulocytic differentiation induced by DMSO was accompanied by an increase of p18INK4C and p19INK4D expression only. PMA treatment of blast cells from two AML patients confirmed these cell line results. Disparate regulation of p15INK4B and p18INK4C mRNA was dependent on intermediary protein synthesis and occurred at the post-transcriptional level as shown by nuclear run-on analysis and mRNA half-life studies. In normal granulocytes and monocytes low constitutive p15INK4B and p18INK4C mRNA expression was detectable by RT-PCR only, but p19INK4D transcripts were noted by Northern blotting in both cell types. Disparate expression of G1-specific cell cycle inhibitors indicates complex and divergent roles of particular CKIs during normal and leukemic myeloid hematopoiesis.

Marrow fibrosis, indicator of therapy failure in chronic myeloid leukemia - prospective long-term results from a randomized-controlled trial.

Marrow fibrosis (MF) has rarely been considered in therapy studies on chronic myeloid leukemia (CML), and there is a lack of long-term observations on the basis of sequential bone marrow biopsies (BMBs) taken prospectively during the course of disease. A total of 848 BMBs from 400 patients with Ph(+) CML recruited in the German randomized CML study I were examined for MF before and during therapy. In total, 110 patients had been randomized to receive interferon (IFN)-alpha, and 290 to receive chemotherapy (hydroxyurea (HU): 154, busulfan: 136). During IFN-alpha and HU medication, MF was reduced or did not increase for about 2 years. Evolving or progressive MF was an independent and early predictor of therapy failure about 2 years earlier than indicated by changes in the peripheral blood, spleen size, marrow blast count and cytogenetics (P<0.00005), resulting in a significant shortening of the survival times of patients independent of the type of therapy applied including allografting (multivariate analyses; P<0.00005). The analyzed long-term observations strongly indicate that MF is an independent poor prognostic complication of CML, allowing an early prediction of therapy failure. Consideration of the fiber content in marrow may therefore significantly improve the prediction of therapy efficacy and outcome of disease.

Treatment intensity significantly influencing fibrosis in bone marrow independently of the cytogenetic response: meta-analysis of the long-term results from two prospective controlled trials on chronic myeloid leukemia.

Bone marrow fibrosis (MF) has been shown to indicate therapy failure in Ph(+) chronic myeloid leukemia (CML). However, the results on the development of MF during interferon-alpha therapy of CML are controversial. The significance of the interferon dose has not been considered as yet. In total, 627 bone marrow biopsies taken prospectively from 200 patients with CML recruited in two studies using different doses of interferon-alpha +/- low-dose cytosine arabinoside were examined for MF before and during therapy. The results showed that the risk of MF depended significantly on the interferon-alpha dose applied (P<0.000005). MF progressed during low-dose therapy (3 x 5 x 10(6) IU/week), but was prevented from progression when applying high dose (5 x 10(6) IU/m(2)/per day). MF disappeared when high-dose interferon-alpha was combined with low-dose cytosine arabinoside (P<0.000005). The risk of death markedly increased when MF occurred or progressed (P<0.0009), independent of all other prognostic factors evaluated including the cytogenetic response. In conclusion, the effectiveness of interferon-alpha on MF depends on the treatment intensity. MF reverses when combining high-dose interferon-alpha with low-dose cytosine arabinoside, but progresses when applying low-dose interferon-alpha. MF appears to be a significant early indicator of ineffective therapy in CML.

Randomized studies with interferon in chronic myelogenous leukemia (CML) and comparative molecular aspects. German CML Study Group.

Four randomized prospective studies on interferon alpha (IFN) in CML report varying degrees of prolongation of the chronic phase of CML and of survival as compared to conventional therapies. There is agreement that IFN prolongs survival as compared to standard busulfan. There is disagreement, however, as to which degree IFN is superior to hydroxyurea. Whereas the randomized studies of the Italian cooperative group and of the British MRC find a statistically significant survival advantage of IFN over hydroxyurea of about 20 months, this difference is only 10 months in the German randomized study and not significant. One reason for this difference might be the more intensive treatment schedule for the hydroxyurea control group in the German study. Other reasons might be differences in risk profiles between the patient groups studied and in strategies of IFN therapy. About 1% of the human genome consists of retroviral or retroviral-like sequences. By analogy to animal models, endogenous retroviruses might also have pathogenic potential in human disease. The transposon-like structure of retroviruses that enables them to integrate at almost any position in the host genome and the capability of retroviruses to serve as efficient vehicles of cellular genes are in support of a pathogenic potential. Furthermore, particles resembling retroviruses have been observed long ago in human embryonic and malignant tissues and cell lines. Sequence information and the transcriptional activity of the endogenous sequences argue against the possibility that these sequences are only fossil relics of early evolutionary periods. Most of the sequences appear to be inactivated by stop codons or frameshifts, making the genomic localization of open reading frames with biological activity difficult. Up to now, mutagenesis by insertion of retroviral-like sequences in sporadic cases of human disease appears to be the only example of pathogenic relevance of retroviruses in man.