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Erythropoiesis is a multistep process involving a large number of genes, which balance between proliferation, differentiation and survival of the erythroid cells. To understand the molecular mechanisms of erythropoiesis and related pathological aberrations, we analyzed three stages of in vitro differentiating human erythroid cells by expression profiling. We identified distinct clusters of genes, each with a unique expression pattern during differentiation. As JAK2 was shown to play a central role in myeloproliferative disorders, we focused on one cluster which includes JAK2 and other genes with high correlation to JAK2 expression. These genes had a low expression at the early erythroblast which increased in the intermediate stage and further slightly increased in the last stage of differentiation. Our results indicate that gene networks may associate with JAK2 expression in erythroid differentiation. It is intriguing to determine whether the pathogenesis of polycythemia vera (PV), harboring a common or uncommon JAK2 mutation, involves alterations in independent gene pathways that underlie the normal erythropoietic process.

The p53 tumor suppressor gene was found to play a role in the differentiation of several tissue types. We report here that p53-dependent apoptosis plays a role in the final stages of physiological differentiation of normoblasts, resulting in nuclear condensation and expulsion without cell death. Blood samples of healthy newborns, cord blood as well as bone marrow, were analysed for apoptosis by TUNEL and p53 expression by immunostaining. While some samples exhibited simultaneously several distinct patterns of apoptosis, such as perinuclear, diffused nuclear or nuclear apoptotic bodies, others presented a single defined pattern. Overexpression of p53 protein was detected in normoblasts exhibiting either perinuclear or diffused nuclear p53, corresponding to the nuclear apoptotic pattern in the same sample. Similar results were also evident with colonies cultivated for 12-14 days in culture. Differentiated erythroid colonies exhibited overexpression of p53 and positive TUNEL staining only in the normoblasts. We further examined the state of caspase 3/7 and observed a decrease of this activated enzyme during erythroid differentiation in culture. This study suggests a novel role for apoptosis in normoblast differentiation where nuclear degradation occurs with a delay in the actual cell death. A pivotal role for the p53-dependent apoptosis in the erythroid lineage development is implied. However, this apoptotic process is not fully executed because of the exhaustion in caspase 3/7 and thus cells are diverted towards final stages of differentiation.

Inactivation of the wild-type p53 gene (TP53) by various genetic alterations is a major event in human tumorigenesis. More than 60% of human primary tumors exhibit a mutation in the p53 gene. Hematological malignancies present a rather low incidence of genetic alterations in this gene (10-20%). Nevertheless, epidemiological studies of the hematological malignancies indicate that the prognosis of patients with a mutation in the p53 gene is worse than those expressing the wild-type p53 protein. Correlations between drug resistance, altered apoptosis, and mutations in the p53 gene are found in hematological malignancies and leukemias. These issues, as well as the possibility of exploiting p53 and its various functions for new therapeutic strategies, are discussed in the present review.

Downregulation of apoptosis has been proposed as a mechanism of clonal expansion in low-grade B cell neoplasms. We have previously described an unusual case of CD5+ B cell lymphoma characterized by cycles of leukemic phase alternating with spontaneous remission. In the present study, we examined the involvement of apoptosis-related proteins in the progression of this cyclic lymphoma ex vivo. During the leukemic phases, the clonal cells were activated blasts expressing elevated levels of wild-type (wt) p53, Bcl-2, Bcl-xL, and Bax, while Bak expression increased during the decline of lymphocytosis. Bax heterodimerized with Bcl-2 but not with Bcl-xL. The anti-apoptotic Bcl-2/Bax heterodimers peaked during early leukemic phases and declined during regression. The elevation in Bcl-2, Bcl-xL and Bax expression during early leukemic phases seems to result from cell activation since a similar increase was induced by activating the remission phase leukemic cells in culture. The data suggest that wt p53, Bcl-xL, and Bcl-2/Bax heterodimers support the accumulation of activated leukemic cells during the leukemic phases, while Bax and Bak may be involved in their decline during regression.

The TP53 gene has been extensively studied in patients with chronic myeloid leukemia (CML), both in chronic phase and in blast crisis. Mutations in the gene were found in up to 30% of the patients, especially among those in blast crisis. We report the results of an analysis of 29 blood samples from CML patients: 8 samples from chronic phase patients, 8 from patients in the accelerated phase, and 13 from patients in blast crisis. By using genomic DNA, we sequenced PCR products of the coding exons and most introns of the TP53 gene, finding genetic changes in 30% of the blast crisis samples and 12% in chronic phase. All mutations were found in introns and were previously unreported. Immunocytochemical studies revealed accumulation of TP53 in blood cells of samples both from chronic phase and blast crisis patients. Since these samples had no TP53 mutations, we believe that wild type TP53 accumulates in blood cells of CML patients. Our results, therefore, indicate that molecular changes in coding regions of the TP53 gene are rare. The significance of the abundance of intronic changes should be investigated further. Accumulation of wild type TP53 in CML cells may indicate an additional mechanism involving this gene in the pathogenesis of this

We present a novel polymorphic 8-bp sequence in intron 6 of the p53 gene that maps between bp 55 and 62 of the 3' end of exon 6. Of normal blood samples, 32% were heterozygotic for this polymorphism and display a NN' genotype, whereas 68% of the population is homozygotic for the N genotype. The rare homozygotic genotype N' was detected only in four blood samples of cancer patients. Peripheral blood of gastrointestinal (GI) and breast tumor patients demonstrated a higher incidence of heterozygosity (50%) than that of normal individuals. Analysis of the distribution of this polymorphism in tumor samples showed loss of heterozygosity (LOH). This LOH during tumor progression could exhibit preference to each one of the polymorphic alleles. The rare presentation of one allele and the increased incidence of heterozygosity in carcinoma patients may suggest an association between this polymorphism with cancer predisposition and susceptibility. The fact that genetic alterations occurring in noncoding regions may play a role in tumor development only further increases the extent of involvement of p53 in carcinogenesis.

To determine the effect of perioperative blood transfusion on immunological parameters, T cells, T-cell subsets, and concanavalin A-induced suppression were measured in 25 patients with colorectal and breast cancer. During the operation, 15 patients received autologous blood and 10 patients had homologous transfusion. The immunological status was again determined after curative surgery. Before surgery, normal percentage of T lymphocytes, decreased ratios of helper/suppressor cells, and impaired con A-induced suppression were found. Following the operation, the helper and suppressor cell percentages reversed to normal, whereas the con A-induced suppression remained impaired. This change was significantly more pronounced in patients who received autologous blood transfusion than in the other group. Autotransfusion has an impact on immune parameters that might prove less detrimental to the clinical outcome in oncologic surgery than homologous transfusion.

p53 inactivation is a key factor in human tumorigenesis and chemotherapy resistance. The traditionally described mechanisms of p53 inactivation in acute myeloid leukemia (AML) include TP53 mutations and abrogation of p53 pathway. Malfunction of wild-type (wt) p53, due to its cytoplasmic mislocalization, has been described, thus far, only in solid tumors. Herein, we present a patient with therapy-related resistant AML, monosomal karyotype, wt TP53, and cytoplasmic sequestration of p53 protein. Proposed mechanisms of p53 mislocalization and their probable clinical and therapeutic implications are discussed. In view of the relative rareness of TP53 mutations in AML, the cytoplasmic sequestration of p53 protein offers an additional inactivating mechanism, which might be more frequent than currently diagnosed. This notion warrants confirmation by prospective studies in large cohorts of patients. We recommend that evaluation of p53 subcellular localization and function should be included in the diagnostic work-up of AML with wt p53.

Large granular lymphocyte disorder (LGLD) is a lymphoproliferative disease, characterized by moderate lymphocytosis with an excess of large granular lymphocytes, neutropenia, anemia, and a variable, but mostly chronic clinical course. We describe two patients with LGLD. One patient presented with symptomatic corticoster-oid-responsive hemolytic anemia, while the other had a chronic course not requiring therapy. The majority of lymphocytes from both patients were CD8 + T lymphocytes. However, the cells from the two patients differed in the molecular pattern of the T cell receptor (TCR), and this may explain the difference in their clinical course.

Normal human monocytes and macrophages generate potent procoagulant activity (PCA) resembling tissue factor (TF) in response to various stimuli. In this study we show that tuftsin, a natural stimulator of many functions of monocytes and macrophages, also stimulates a potent PCA in mixed mononuclear cells and monocytes, and a mild PCA in lymphocytes and cell lines of monocytic origin (U937 and THP). No activity was generated by several lymphoid cell lines and HL-60 cells. The PCA resembled TF in that it accelerated clotting through the extrinsic coagulation pathway and was inhibited by concanavalin-A and by monoclonal anti-TF antibodies. The induction of TF-like activity by tuftsin was dose- and time-dependent. It was located in the cell membrane and did not require T cells for expression. Generation of TF- like activity was prevented by actinomycin D, while cytarabine had no effect on this process, suggesting that expression of the activity depends on protein synthesis. Studies with various tuftsin analogs suggest that tuftsin stimulates generation of TF-like activity, as well as other functions of monocytes via the same receptors. The results with the monocytic cell lines show that tuftsin affects mainly mature cells. The induction of TF-like activity in mononuclear cells by tuftsin constitutes an important link between mononuclear cells and the immune and coagulation systems. It may play a major role in the pathogenesis of thromboembolism and fibrin deposition in various inflammatory and immunologic disorders.

LCC2, an estradiol-independent tamoxifen (Tax)-resistant subline of MCF-7 human breast cancer cell line, is resistant relatively towards Tax and methotrexate (Mtx). The purpose of the present study is to evaluate the role of p53 in determining this resistance. While MCF-7 is sensitive to and undergoes apoptosis, as determined by propidium iodide stain, by Tax and Mtx, LCC2 is resistant to apoptosis induction by these agents. Both cell lines undergo apoptosis and are sensitive equally to doxorubicin (Adr). p53 cDNA of both sublines was evaluated by polymerase chain reaction (PCR) amplification and sequencing and was found to be of wild-type. p53 mRNA, as well as protein, are elevated markedly in LCC2 as compared to MCF-7 cells. p53 expression was increased by estradiol and Adr, not changed by Mtx, and decreased by Tax and estradiol-deprivation in both sublines. p53 modulation by the various agents, in both sublines, was evaluated by cytochemical staining and subcellular fractionation. This analysis showed that p53 is localized mainly in the nuclear fraction in MCF-7 cells, and in the cytoplasmatic fraction in LCC2 cells. Doxorubicin induced apoptosis in MCF-7 cells along with increase in its nuclear fraction. In contrast, LCC2 underwent apoptosis by Adr despite its cytoplasmatic sequestration. These experiments demonstrate that p53 is sequestered to cytoplasm in the estrogen-independent, Tax-resistant LCC2 cells. However, the differences in apoptotic rate between MCF-7 and LCC2 cells do not seem to be dependent on p53. The LCC2 cell line may serve as a useful model for the study of the mechanism of cytoplasmatic sequestration of wild type (wt) p53, its physiologic consequences, and its relation to estrogen-independence or Tax resistance of breast cancer cells.

The p53 tumor suppressor gene is frequently mutated in most human malignancies. These mutations have been associated with clinical outcome for various cancer types. Since this gene's main function is to guard the integrity of the genome, its clinical relevance, i.e. its role in carcinogenesis and chemotherapy-drug resistance, have been extensively studied. These data and the p53 protein as a potential target for new treatment strategies, are reviewed based on acquired knowledge of the structure–function of the p53 protein.

A human T-acute lymphoblastic leukemia (ALL) cell line (Loucy), derived from cells from a patient with resistant ALL with a t(16:20) and 5q- chromosomal aberrations was evaluated for p53 gene alterations and expression. Western blot analysis of p53 showed elevated levels of the protein. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and direct sequencing identified a point mutation at codon 272 (GTG --> ATG) of the p53 gene. Possible molecular mechanisms underlying these alterations and their role in the establishment of this cell line and in leukemogenesis in general are discussed.

Mutations in thep53 tumor suppressor gene have been found to be the most frequent genetic alterations in human malignancies. To further examine the idea that neoplastic progression is associated with mutations in the p53 gene, we analyzed matched primary and metastatic tumor samples. The samples included 15 pairs of breast cancer and metastases to lymph nodes, four pairs of gastrointestinal adenocarcinomas and metastases to liver, one colon adenocarcinoma and metastasis to a lymph node, and one lung carcinoma and metastasis in the pleura. Genomic DNA or cDNA from each tumor sample was amplified by the polymerase chain reaction and labeled by using one biotinylated primer. The DNA strands were separated with magnetic streptavidin beads and sequenced directly. p53 mutations were detected in 11 of 21 patients (52%) in either primary tumors, metastases, or both. In six of these patients the primary tumor and matched metastasis shared the same single mutation. In the other patients an additional mutation in the primary tumor only or a mutation in the metastasis only was observed. Our data suggest that tumor development and progression toward metastasis involves structural alterations in the ps3 gene that occur early in carcinogenesis. In some cases, genetic changes in metastatic spreading may also include the appearance of a mutation in a metastasis derived from a primary tumor expressing wild-type p53, a selection of metastatic cells with a single mutation from a primary tumor expressing two different mutations, or loss of heterozygosity. © 1995 Wiley-Liss, Inc.

Neutrophils from cord blood of healthy term infants were isolated and incubated for 30 min with varying concentrations of intravenous lipid emulsion (ILE) solution (4, 8, 20 mg/ml). In vitro assay of chemotaxis was performed after incubation for 120 min with endotoxin-activated serum (EAS). Neutrophil random motility was unchanged after ILE incubation yet chemotactic factor (EAS)-stimulated motility was significantly reduced in a dose-related pattern. (Journal of Parenteral and Enteral Nutrition14:472-473, 1990)

Endometrial lymphocytes were isolated in two groups of fertile women, 17 of them normal and seven with various clinical problems. In order to study their immunological properties, endometrial and blood lymphocytes were cultured with either phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed (PW), or purified protein derivative (PPD). Lymphocyte response was assayed by tritiated thymidine incorporation. The percent of T cells was estimated by E-rosette formation. In the normal fertile group, endometrial lymphocyte response to PHA and to Con A was found to correlate with the days of menstrual cycle, rising in a linear fashion between days 9 and 24. In the group with clinical problems, no correlation was noted. In neither group were endometrial lymphocyte responses to PW or PPD or percent of endometrial lymphocyte rosette formation influenced by the menstrual cycle. The modulation of endometrial lymphocyte response to PHA or to Con A stimulation in normal fertile women during the menstrual cycle seems unrelated to the number of T cells.

PMN leukocytes from untreated patients with multiple myeloma (MM), Hodgkin's disease (HD) and non-Hodgkin lymphoma (NHL) were studied in vitro for their phagocytic and chemotactic function. Alkaline phosphatase score and the reduction of nitroblue tetrazolium (NBT) in these leukocytes were also determined. Most of the functions of PMN leukocytes from untreated patients with MM were impaired, compared to control leukocytes, while those from patients with HD and NHL were impaired only in their chemotactic response to casein and endotoxin-activated serum (EAS).

Molecular structural analysis of the p53 gene in patients with Philadelphia chromosome-positive chronic myelogenous leukemia (CML) indicates a significant incidence of gene rearrangements in patients at either accelerated phase or blastic crisis. Southern blot analysis of genomic DNA hybridizing with either genomic or cDNA p53 specific probes indicated that 30% of the CML patients at blastic crisis phase exhibited rearrangements, mostly mapping downstream to the first non- coding exon. This is compatible with the observation that the progression of CML from the chronic to the acute phase involves frequent aberrations in chromosome 17, to which the p53 oncogene has been mapped. Therefore, we suggest that one of the pathways of development of CML to the acute phase is associated with aberrations in the p53 nuclear oncogene.

Various immunologic parameters were tested in a family of two patients with common variable immunodeficiency. Both patients had low serum immunoglobulin levels, low peripheral B cell and T4 subclass of lymphocytes, and reversed T4T8 ratio. One of the patients had excessive suppressor activity. Two of the asymptomatic members of the family (the father and one brother) had also low T4T8 ratio that was not associated with excessive suppressor activity. No linkage between the disease inheritance to HLA could be observed. A study of the T cell helper activity to an antigen, the response to which is regulated by an HLA-linked gene, suggested a defect in the immune response of the two patients and their asymptomatic brother with immunologic disorders. Treatment with cimetidine of the patient with excessive suppressor activity led to an improvement in his clinical state, reduction in suppressor activity, temporary effect on his proliferative response capacity to mitogens, and an increase in the antigen-specific helper activity.