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Patients with acute promyelocytic leukemia (APL) usually relapse after all-trans retinoic acid (RA) treatment because this therapy fails to eradicate the malignant clone. Our data showed that KH 1060 and other 20-epi vitamin D3 analogs alone were potent inhibitors of clonal growth of NB4 cells, an APL cell line (ED50, approximately 5 x 10(-11) M). The combination of KH 1060 and 9-cis-RA synergistically and irreversibly enhanced this effect. Neither KH 1060 nor 9-cis-RA (10(-6) M, 3 d) were strong inducers of differentiation of NB4 cells. However, 98% of the cells underwent differentiation to a mature phenotype with features of both granulocytes and monocytes after exposure to a combination of both compounds. Apoptosis only increased after incubation of NB4 cells with 9-cis-RA alone (28%) or with a combination of 9-cis-RA plus KH1060 (32%). Immunohistochemistry showed that the bcl-2 protein decreased from nearly 100% of the wild-type NB4 cells to 2% after incubation with a combination of KH 1060 and 9-cis-RA, and the bax protein increased from 50% of wild-type NB4 cells to 92% after culture with both analogs (5 x 10(-7) M, 3 d). Western blot analysis paralleled these results. Studies of APL cells from one untreated individual paralleled our results with NB4 cells. Taken together, the data demonstrated that nearly all of the NB4 cells can be irreversibly induced to differentiate terminally when exposed to the combination of KH 1060 and 9-cis-RA.

Post-transplant lymphoproliferative disorder (PTLD) is a complication of allogeneic bone marrow transplantation (BMT). Rare cases of PTLD after autologous BMT have been reported only in adults. This case report is the first to describe PTLD in a pediatric patient after autologous peripheral stem cell transplantation (PSCT). This 2-year-old male with stage IV neuroblastoma underwent autologous PSCT. The post-PSCT course was complicated by fever with hematochezia and a lung mass. On day 94 post PSCT, colonoscopy revealed an ulcer due to a PTLD, monomorphic type, B cell phenotype, associated with Epstein–Barr virus. Fine needle aspiration identified the lung mass as neuroblastoma. PTLD can occur in pediatric autologous PSCT recipients, and may occur more frequently in autologous grafts manipulated by T cell depletion or CD34+ cell selection. Bone Marrow Transplantation (2000) 26, 1021–1024. [ABSTRACT FROM AUTHOR]

Prostate stem cell antigen (PSCA) is a recently defined homologue of the Thy-1/Ly-6 family of glycosylphosphatidylinositol (GPI)-anchored cell surface antigens. PSCA mRNA is expressed in the basal cells of normal prostate and in more than 80% of prostate cancers. The purpose of the present study was to examine PSCA protein expression in clinical specimens of human prostate cancer. Five monoclonal antibodies were raised against a PSCA-GST fusion protein and screened for their ability to recognize PSCA on the cell surface of human prostate cancer cells. Immunohistochemical analysis of PSCA expression was performed on paraffin-embedded sections from 25 normal tissues, 112 primary prostate cancers and nine prostate cancers metastatic to bone. The level of PSCA expression in prostate tumors was quantified and compared with expression in adjacent normal glands. The antibodies detect PSCA expression on the cell surface of normal and malignant prostate cells and distinguish three extracellular epitopes on PSCA. Prostate and transitional epithelium reacted strongly with PSCA. PSCA staining was also seen in placental trophoblasts, renal collecting ducts and neuroendocrine cells in the stomach and colon. All other normal tissues tested were negative. PSCA protein expression was identified in 105/112 (94%) primary prostate tumors and 9/9 (100%) bone metastases. The level of PSCA expression increased with higher Gleason score (P=0.016), higher tumor stage (P=0.010) and progression to androgen-independence (P=0.021). Intense, homogeneous staining was seen in all nine bone metastases. PSCA is a cell surface protein with limited expression in extraprostatic normal tissues. PSCA expression correlates with tumor stage, grade and androgen independence and may have prognostic utility. Because expression on the surface of prostate cancer cells increases with tumor progression, PSCA may be a useful molecular target in advanced prostate cancer. Oncogene (2000) 19, 1288–1296. [ABSTRACT FROM AUTHOR]

The central hypothesis underlying specific anti-leukemia immunotherapy is that leukemic cells express antigenic determinants not expressed on their counterpart normal adult cells. We have developed a murine myeloid leukemia/tumor immunization model using the low-immunogenic WEHI3 leukemia in syngeneic mice. Mice preimmunized with irradiated, transduced IL-7-producing WEHI3 cells showed systemic protection and rejection of a lethal dose of intravenously (i.v.) injected parental WEHI3 cells (5 x 10(4)) with 40% long-term survival. When vaccinated with a mixture of parental WEHI3 cells and IL-2-producing NIH-3T3 fibroblasts (5 x 10(5)), 60% survival was observed. Vaccination with murine granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing WEHI3 cells resulted in only 20% survival of i.v. challenged mice, and the additional combination of IL-2- and IL-7-producing vaccine did not reveal any additive or synergistic effects. Immunizing mice with a pre-established leukemia burden (injected with 5 x 10(4) WEHI3 cells, i.v., 3 days prior to immunization) did not cure or result in a prolongation of survival, indicating that improved methods of immunization are needed. Taken together, we have identified IL-7 and IL-2 as effective cytokines in our leukemia/vaccination model with only marginal activity by GM-CSF. [ABSTRACT FROM AUTHOR]

An imbalance between cellular apoptosis and survival may be critical for the pathogenesis of lymphoma. Therefore, the gene expression pattern in lymph node preparations from patients with mantle cell lymphoma (MCL) was compared to the pattern in nonmalignant hyperplastic lymph nodes (HLs). Oligonucleotide microarray analysis was performed comparing 5 MCLs to 4 HLs using high-density microarrays. The expression data were analyzed using Genespring software. For confirmation, the expression of selected genes was analyzed by real-time polymerase chain reaction using the RNA extracted from 16 MCL and 12 HL samples. The focus was on 42 genes that were at least 3-fold down-regulated in MCL; in addition to the B-cell leukemia 2 (BCL2) system other apoptotic pathways were altered in MCL. The FAS-associated via death domain (FADD) gene that acts downstream of the FAS cascade as a key gene to induce apoptosis was more than 10-fold down-regulated in MCL. Furthermore, the death-associated protein 6 (DAXX) gene, the caspase 2 (CASP2) gene, and the RIPK1 domain containing adapter with death domain (RAIDD) gene, which are key genes in other proapoptotic pathways, were also decreased in the MCL samples. The suggestion is made that in addition to the known overexpression of cyclin D1, which drives entry into the cell cycle, disturbances of pathways associated with apoptosis contribute to the development of MCL. (Blood. 2001;98:787-794)

The CHK2 gene encodes a protein kinase that is important for the regulation of cell cycle arrest after DNA damage. CHK2 acts downstream of ataxia teleangiecstasia mutated (ATM), modulates the function of p53 and may help mediate cell cycle arrest at G2/M by phosphorylation of Cdc25C. Recently, the human homolog of the checkpoint kinase Cds1 (CHK2) has been suggested to be a tumor suppressor gene. Heterozygous germline mutations have been reported in Li-Fraumeni syndrome (LFS), a highly penetrant familial cancer phenotype, and in sporadic colon cancer. LFS is associated with the development of lymphoid malignancies, especially childhood ALL. Therefore, we analyzed the DNA from 143 lymphoid malignancies to determine whether they had mutations of the CHK2 gene. The 14 exons of CHK2 were studied by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and sequencing of aberrantly migrating bands. One missense mutation changing serine to phenylalanine (codon 428) in an evolutionarily highly conserved domain was found in a non-Hodgkin's aggressive lymphoma. Another point mutation in the non-coding region was identified in one of adult T-cell leukemias (ATL) samples. This result suggests that mutation of the CHK2 gene may rarely be involved in the development of selected lymphomas.

Purpose: We analyzed the effects of the change in TNM classification from the 1987 to the 1997 version and suggest a modified tumor size cutoff point between T stages 1 and 2 for renal cell carcinoma., Materials and Methods: We evaluated a database containing the records of 661 patients who underwent nephrectomy between 1989 and 1999. The effect of the change in TNM classification on the distribution of patients between stages, the rates of M+ and N+ disease, and the local and distant recurrence rates were outlined for 280 patients with T stages 1 and 2 disease. The Cox model was used to identify the optimal cutoff point between T1 and T2 disease, and the resulting effect of adopting this cutoff was outlined., Results: A total of 174 and 128 cases were down staged from 1987 version stage T2 to 1997 version stage T1 and from 1987 TNM stage II to 1997 TNM stage I, respectively. Survival was not significantly different in patients with 1997 TNM stages I and II disease due to a lack of survival difference during the first 2 years of followup. Stage shift also caused an increase in average tumor size, the proportion of patients with high grade cancer, and M+ and N+ disease at diagnosis in 1997 stages T1 and T2 as well as an increase in the proportion of 1997 stage T2N0M0 cases at diagnosis with systemic failure. Analysis of 11 potential cutoff points between 1 and 10 cm. revealed that 4.5 cm. was most predictive of patients survival (hazards ratio 4.99, p = 0.0001). Using this cutoff resulted in improved discriminatory power of the TNM classification and a moderating effect on the distribution of patients, average tumor size, high grade disease, M+ and N+ disease at diagnosis, and systemic failure between T(14.5) and T(24.5) compared with 1997 T1 and T2., Conclusions: Our data imply that the current cutoff point of 7 cm. between stages T1 and T2 tumors is too high. Lowering the cutoff to 4.5 cm. resulted in better discriminatory power of the TNM classification in our dataset. This observation should be further validated by external data.

Aneuploidy is a characteristic of the majority of human cancers, and recent studies suggest that defects of mitotic checkpoints play a role in carcinogenesis. MAD1L1 is a checkpoint gene, and its dysfunction is associated with chromosomal instability. Rare mutations of this gene have been reported in colon and lung cancers. We examined a total of 44 cell lines (hematopoietic, prostate, osteosarcoma, breast, glioblastoma and lung) and 133 fresh cancer cells (hematopoietic, prostate, breast and glioblastoma) for alterations of MAD1L1 by RT-PCR-SSCP and nucleotide sequencing. Eight mutations consisting of missense, nonsense and frameshift mutations were found, together with a number of nucleotide polymorphisms. All the alterations in cell lines were heterozygous. Frequency of mutations was relatively high in prostate cancer (2/7 cell lines and 2/33 tumor specimens). We placed a mutant truncated MAD1L1, found in a lymphoma sample, into HOS, Ht161 and SJSA cell lines and found that it was less inhibitory than wild type MAD1L1 at decreasing cell proliferation. Co-expression experiments showed that the mutant form had a dominant-negative effect. Furthermore, this mutant impaired the mitotic checkpoint as shown by decreased mitotic indices in HOS cells expressing mutant MAD1L1 after culture with the microtubule-disrupting agent, nocodazole. Our results suggest a pathogenic role of MAD1L1 mutations in various types of human cancer.

Activation of the TCL1 oncogene has been implicated in T cell leukemias/lymphomas and recently was associated with AIDS diffuse large B cell lymphomas (AIDS-DLBCL). Also, in nonmalignant lymphoid tissues, antibody staining has shown that mantle zone B cells expressed abundant Tcl1 protein, whereas germinal center (GC; centrocytes and centroblasts) B cells showed markedly reduced expression. Here, we analyze isolated B cell subsets from hyperplastic tonsil to determine a more precise pattern of Tcl1 expression with development. We also examine multiple B cell lines and B lymphoma patient samples to determine whether different tumor classes retain or alter the developmental pattern of expression. We show that TCL1 expression is not affected by Epstein-Barr virus (EBV) infection and is high in naïve B cells, reduced in GC B cells, and absent in memory B cells and plasma cells. Human herpesvirus-8 infected primary effusion lymphomas (PEL) and multiple myelomas are uniformly TCL1 negative, whereas all other transformed B cell lines tested express moderate to abundant TCL1. This observation supports the hypothesis that PEL, like myeloma, usually arise from post-GC stages of B cell development. Tcl1 protein is also detected in most naïve/GC-derived B lymphoma patient samples (23 of 27 [85%] positive), whereas most post-GC-derived B lymphomas lack expression (10 of 41 [24%] positive). These data indicate that the pattern of Tcl1 expression is distinct between naïve/GC and post-GC-derived B lymphomas (P < 0.001) and that the developmental pattern of expression is largely retained. However, post-GC-derived AIDS-DLBCL express TCL1 at a frequency equivalent to naïve/GC-derived B lymphomas in immune-competent individuals (7 of 9 [78%] positive), suggesting that TCL1 down-regulation is adversely affected by severe immune system dysfunction. These findings demonstrate that TCL1 expression in B cell lymphoma usually reflects the stage of B cell development from which they derive, except in AIDS-related lymphomas.

Purpose: To integrate stage, grade, and Eastern Cooperative Oncology Group (ECOG) performance status (PS) into a clinically useful tool capable of stratifying the survival of renal cell carcinoma (RCC) patients., Patients and Methods: The medical records of 661 patients undergoing nephrectomy at University of California Los Angeles between 1989 and 1999 were evaluated. Median age was 61 years, male-to-female ratio was 2.2:1, and median follow-up was 37 months. Survival time was the primary end point assessed. Sixty-four possible combinations of stage, grade, and ECOG PS were analyzed and collapsed into distinct groups. The internal validity of the categorized was challenged by a univariate analysis and a multivariate analysis testing for the accountability of each UCLA Integrated Staging System (UISS) category against independent variables shown to have impact on survival., Results: Combining and stratifying 1997 tumor-node-metastasis stage, Fuhrman's grade and ECOG PS resulted in five survival stratification groups designated UISS, and numbered I to V. The projected 2- and 5-year survival for the UISS groups are as follows for the groups: I, 96% and 94%; II, 89% and 67%; III, 66% and 39%; IV, 42% and 23%; and V, 9% and 0%, respectively. UISS accounted for the significant variables in the variate analysis., Conclusion: A novel system for staging and predicting survival for RCC integrating clinical variables is offered. UISS is simple to use and is superior to stage alone in differentiating patients' survival. Our data suggests that UISS is an important prognostic tool for counseling patients with various stages of kidney cancer. Further prospective large-scale validation with external data is awaited.

Prostatic small cell carcinoma is an aggressive subtype of prostate cancer that usually appears as a progression of the original adenocarcinoma. We describe here the WISH-PC2, a novel neuroendocrine xenograft of small cell carcinoma of the prostate. This xenograft was established from a poorly differentiated prostate adenocarcinoma and is serially transplanted in immune-compromised mice where it grows within the prostate, liver, and bone, inducing osteolytic lesions with foci of osteoblastic activity. It secretes to the mouse Chromogranin A and expresses prostate plasma carcinoma tumor antigen-1, six-transmembrane epithelial antigen of the prostate, and members of the Erb-B receptor family. It does not express prostate-specific antigen, prostate stem cell antigen, prostate-specific membrane antigen, and androgen receptor, and it grows independently of androgen. Altogether, WISH-PC2 provides an unlimited source in which to study the involvement of neuroendocrine cells in the progression of prostatic adenocarcinoma and can serve as a novel model for the testing of new therapeutic strategies for prostatic small cell carcinoma.

Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus, has been implicated in the pathogenesis of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), multicentric Castleman's disease, and recently multiple myeloma (MM). DNA sequence analyses of HHV-8 suggest that multiple HHV-8 strains exist. We extracted DNA from 24 patients with MM and 3 patients with monoclonal gammopathy of undetermined significance and compared HHV-8 open reading frames (ORFs) 26 and 65 sequences with those derived from patients with KS, PEL, and two HHV-8-positive PEL cell lines KS-1 and BC-1. ORF26 sequence data suggest that MM patients are consistently carriers of HHV-8 strain subtype C3. All MM patients also consistently revealed either a single bp deletion or substitution at position 112197 in ORF65. This unique alteration is not present in patients with KS or PEL or in PEL cell lines. It occurs in the portion of ORF65 that is known to be responsible for a serological response to HHV-8.

In most cells, transferrin receptor (TfR1)-mediated endocytosis is a major pathway for cellular iron uptake. We recently cloned the human transferrin receptor 2 (TfR2) gene, which encodes a second receptor for transferrin (Kawabata, H., Yang, R., Hirama, T., Vuong, P. T., Kawano, S., Gombart, A. F., and Koeffler, H. P. (1999) J. Biol. Chem. 274, 20826-20832). In the present study, the regulation of TfR2 expression and function was investigated. A select Chinese hamster ovary (CHO)-TRVb cell line that does not express either TfR1 or TfR2 was stably transfected with either TfR1 or TfR2-alpha cDNA. TfR2-alpha-expressing cells had considerably lower affinity for holotransferrin when compared with TfR1-expressing CHO cells. Interestingly, in contrast to TfR1, expression of TfR2 mRNA in K562 cells was not up-regulated by desferrioxamine (DFO), a cell membrane-permeable iron chelator. In MG63 cells, expression of TfR2 mRNA was regulated in the cell cycle with the highest expression in late G(1) phase and no expression in G(0)/G(1). DFO reduced cell proliferation and DNA synthesis of CHO-TRVb control cells, whereas it had little effect on TfR2-alpha-expressing CHO cells when measured by clonogenic and cell cycle analysis. In addition, CHO cells that express TfR2-alpha developed into tumors in nude mice whereas CHO control cells did not. In conclusion, TfR2 expression may be regulated by the cell cycle rather than cellular iron status and may support cell growth both in vitro and in vivo.

Background: Management of prostate cancer that has spread beyond the capsule is a difficult problem. Innovative and nontoxic approaches to the disease are urgently required. Recently, a commercially available herbal mixture called PC-SPES showed potent antitumor activities on a variety of malignant cells in vitro., Methods: PC-SPES was evaluated for its ability to inhibit clonal growth, and to induce cell cycle arrest of three human prostate cancer cell lines (LNCaP, PC-3, and DU 145). Western blot analysis examined the effect of PC-SPES on levels of p21(waf1), p27(kip1), Bcl-2, and E-cadherin in the three cell lines; and telomerase activity was examined by telomeric repeat amplification protocol (TRAP) assay. Furthermore, the effect of oral PC-SPES (250 mg/kg/day) on growth of PC-3 and DU 145 tumors present in male BNX nu/nu triple immunodeficient mice was studied. LNCaP cells were not analyzed in mice because they grow only with difficulty in these immunodeficient mice., Results: PC-SPES markedly inhibited clonal growth of LNCaP, PC-3, and DU 145 prostate cancer cells, with a 50% inhibition (ED50) at approximately 2 microl/ml. Pulse-exposure studies showed that a 5-day pulse-exposure to PC-SPES (2 microl/ml) in liquid culture achieved a 50% inhibition of PC-3 clonal growth in soft agar, suggesting that the growth inhibition mediated by the extracts remained after removal of PC-SPES. Cell cycle analysis using the prostate cancer cell lines found that PC-SPES induced a significant increase in the number of cells in G0-G1 and G2/M, with a concomitant decrease in the number of cells in S phase. PC-SPES (2 microl/ml, 4 days) increased slightly the levels of p21(waf1) in the three cell lines, decreased by 40% the levels of Bcl-2 in PC-3, and the levels of p27(kip1) and E-cadherin and telomerase were unchanged in each of the lines. In vivo treatment with oral PC-SPES of male BNX mice having DU 145 tumors produced significant inhibition of their growth (P < 0.001), with no objective side effects including blood chemistries, weights, or autopsy analysis. The PC-SPES showed no statistical effect on the in vivo growth of PC-3 cells., Conclusions: PC-SPES inhibits clonal proliferation of human prostate cancer cells both in vitro and in vivo, using a murine model., (Copyright 2000 Wiley-Liss, Inc.)