Treatment of stage I seminoma, with one course of adjuvant carboplatin or surveillance, risk-adapted recommendations implementing patient autonomy: a report from the Swedish and Norwegian Testicular Cancer Group (SWENOTECA).

BACKGROUND: The purpose of the protocol was to reduce the treatment burden in clinical stage I (CSI) seminoma by offering risk-adapted treatment. The protocol aimed to prospectively validate the proposed risk factors for relapse, stromal invasion of the rete testis and tumor diameter >4 cm, and to evaluate the efficacy of one course of adjuvant carboplatin. PATIENTS AND METHODS: From 2007 to 2010, 897 patients were included in a prospective, population-based, risk-adapted treatment protocol implementing one course of adjuvant carboplatin AUC7 (n = 469) or surveillance (n = 422). In addition, results from 221 patients receiving carboplatin between 2004 and 2007 are reported. RESULTS: At a median follow-up of 5.6 years, 69 relapses have occurred. Stromal invasion of the rete testis [hazard ratio (HR) 1.9, P = 0.011] and tumor diameter >4 cm (HR 2.7, P < 0.001) were identified as risk factors predicting relapse. In patients without risk factors, the relapse rate (RR) was 4.0% for patients managed by surveillance and 2.2% in patients receiving adjuvant carboplatin. In patients with one or two risk factors, the RR was 15.5% in patients managed by surveillance and 9.3% in patients receiving adjuvant carboplatin. We found no increased RR in patients receiving carboplatin <7 × AUC compared with that in patients receiving ≥7 × AUC. CONCLUSION: Stromal invasion in the rete testis and tumor diameter >4 cm are risk factors for relapse in CSI seminoma. Patients without risk factors have a low RR and adjuvant therapy is not justified in these patients. The efficacy of adjuvant carboplatin is relatively low and there is need to explore more effective adjuvant treatment options in patients with high-risk seminoma. The data do not support the concept of a steep dose response for adjuvant carboplatin.

One course of adjuvant BEP in clinical stage I nonseminoma mature and expanded results from the SWENOTECA group.

BACKGROUND: SWENOTECA has since 1998 offered patients with clinical stage I (CS I) nonseminoma, adjuvant chemotherapy with one course of bleomycin, etoposide and cisplatin (BEP). The aim has been to reduce the risk of relapse, sparing patients the need of toxic salvage treatment. Initial results on 312 patients treated with one course of adjuvant BEP, with a median follow-up of 4.5 years, have been previously published. We now report mature and expanded results. PATIENTS AND METHODS: In a prospective, binational, population-based risk-adapted treatment protocol, 517 Norwegian and Swedish patients with CS I nonseminoma received one course of adjuvant BEP. Patients with lymphovascular invasion (LVI) in the primary testicular tumor were recommended one course of adjuvant BEP. Patients without LVI could choose between surveillance and one course of adjuvant BEP. Data for patients receiving one course of BEP are presented in this study. RESULTS: At a median follow-up of 7.9 years, 12 relapses have occurred, all with IGCCC good prognosis. The latest relapse occurred 3.3 years after adjuvant treatment. The relapse rate at 5 years was 3.2% for patients with LVI and 1.6% for patients without LVI. Five-year cause-specific survival was 100%. CONCLUSIONS: The updated and expanded results confirm a low relapse rate following one course of adjuvant BEP in CS I nonseminoma. One course of adjuvant BEP should be considered a standard treatment in CS I nonseminoma with LVI. For patients with CS I nonseminoma without LVI, one course of adjuvant BEP is also a treatment option.

Novel form of collective movement by soil bacteria.

Although migrations are essential for soil microorganisms to exploit scarce and heterogeneously distributed resources, bacterial mobility in soil remains poorly studied due to experimental limitations. In this study, time-lapse images collected using live microscopy techniques captured collective and coordinated groups of B. subtilis cells exhibiting "crowd movement". Groups of B. subtilis cells moved through transparent soil (nafion polymer with particle size resembling sand) toward plant roots and re-arranged dynamically around root tips in the form of elongating and retracting "flocks" resembling collective behaviour usually associated with higher organisms (e.g., bird flocks or fish schools). Genetic analysis reveals B. subtilis flocks are likely driven by the diffusion of extracellular signalling molecules (e.g., chemotaxis, quorum sensing) and may be impacted by the physical obstacles and hydrodynamics encountered in the soil like environment. Our findings advance understanding of bacterial migration through soil matrices and expand known behaviours for coordinated bacterial movement.

Antibody responses after first and second Covid-19 vaccination in patients with chronic lymphocytic leukaemia.

B-cell chronic lymphocytic leukaemia (CLL) is associated with immunosuppression and patients are at increased clinical risk following SARS-CoV-2 infection. Covid-19 vaccines offer the potential for protection against severe infection but relatively little is known regarding the profile of the antibody response following first or second vaccination. We studied spike-specific antibody responses following first and/or second Covid-19 vaccination in 299 patients with CLL compared with healthy donors. 286 patients underwent extended interval (10-12 week) vaccination. 154 patients received the BNT162b2 mRNA vaccine and 145 patients received ChAdOx1. Blood samples were taken either by venepuncture or as dried blood spots on filter paper. Spike-specific antibody responses were detectable in 34% of patients with CLL after one vaccine (n = 267) compared to 94% in healthy donors with antibody titres 104-fold lower in the patient group. Antibody responses increased to 75% after second vaccine (n = 55), compared to 100% in healthy donors, although titres remained lower. Multivariate analysis showed that current treatment with BTK inhibitors or IgA deficiency were independently associated with failure to generate an antibody response after the second vaccine. This work supports the need for optimisation of vaccination strategy in patients with CLL including the potential utility of booster vaccines.

Extracellular, cell-permeable survivin inhibits apoptosis while promoting proliferative and metastatic potential.

The tumour microenvironment is believed to be involved in development, growth, metastasis, and therapy resistance of many cancers. Here we show survivin, a member of the inhibitor of apoptosis protein (IAP) family, implicated in apoptosis inhibition and the regulation of mitosis in cancer cells, exists in a novel extracellular pool in tumour cells. Furthermore, we have constructed stable cell lines that provide the extracellular pool with either wild-type survivin (Surv-WT) or the previously described dominant-negative mutant survivin (Surv-T34A), which has proven pro-apoptotic effects in cancer cells but not in normal proliferating cells. Cancer cells grown in conditioned medium (CM) taken from Surv-WT cells absorbed survivin and experienced enhanced protection against genotoxic stresses. These cells also exhibited an increased replicative and metastatic potential, suggesting that survivin in the tumour microenvironment may be directly associated with malignant progression, further supporting survivin's function in tumourigenesis. Alternatively, cancer cells grown in CM taken from the Surv-T34A cells began to apoptose through a caspase-2- and caspase-9-dependent pathway that was further enhanced by the addition of other chemo- and radiotherapeutic modalities. Together our findings suggest a novel microenvironmental function for survivin in the control of cancer aggressiveness and spread, and should result in the genesis of additional cancer treatment modalities.

Biosynthesis and in vivo localization of the decapentaplegic-Vg-related protein, DVR-6 (bone morphogenetic protein-6).

DVR-6 (BMP-6 or Vgr-1) is a member of the TGF-beta superfamily of polypeptide signaling molecules. In situ hybridization studies have previously shown that DVR-6 RNA is expressed in a variety of cell types in the mouse embryo, but no information has been available on protein localization and biosynthesis. We have produced a polyclonal antibody to the proregion of DVR-6 and used it to localize the protein in whole mount and sectioned embryonic, newborn, and adult mouse tissues. DVR-6 protein is expressed in the mouse nervous system beginning at 9.5 days postcoitum (d.p.c.) and continues through adulthood. A variety of epithelial tissues also produce DVR-6 protein, including the suprabasal layer of the skin, bronchiolar epithelium, and the cornea. Additionally, a stably transfected cell line, BMGE+H/D6c4, is used to study the biosynthesis of DVR-6 protein and evidence is presented for translational regulation of DVR-6 expression.

[Cognitive competence of older workers]. / Kognitive Leistungsfähigkeit älterer Arbeitnehmer.

The article provides a short overview on cognitive strengths and weaknesses of older workers. The older people who are under the age of 65 years already exhibit changes in performance and neurophysiological measures in laboratory tasks that test fluid intelligence. Apart from deficits, the physiological data of the older people show clear evidence for compensatory strategies. Cognitive performance on the current job is usually less impaired. This can be explained by practice, learning, and selection, which are, however, often linked with higher effort. The cognitive competence of older employees varies exorbitantly, which is due to the influence of various factors like education, lifestyle, and in particular the type of work. Thus, the cognitive competence of older employees can be preserved and possibly even enhanced by changes in the work situation as well as by individual training procedures such as cognitive training.

TGF-beta related genes in development.

Embryonic induction is the process by which signals from one cell population change the developmental fate of another. Polypeptides related to growth factors are one group of molecules mediating many inductive events. Recent data on the embryonic expression and function of signaling proteins related to transforming growth factor beta, in both vertebrate and invertebrate systems, have shown that these molecules play important roles in both pattern formation and tissue specification during embryogenesis.

Cancer gene therapy using a survivin mutant adenovirus.

We have constructed a replication-deficient adenovirus encoding a nonphosphorylatable Thr(34)-->Ala mutant of the apoptosis inhibitor survivin (pAd-T34A) to target tumor cell viability in vitro and in vivo. Infection with pAd-T34A caused spontaneous apoptosis in cell lines of breast, cervical, prostate, lung, and colorectal cancer. In contrast, pAd-T34A did not affect cell viability of proliferating normal human cells, including fibroblasts, endothelium, or smooth muscle cells. Infection of tumor cells with pAd-T34A resulted in cytochrome c release from mitochondria, cleavage of approximately 46-kDa upstream caspase-9, processing of caspase-3 to the active subunits of approximately 17 and 19 kDa, and increased caspase-3 catalytic activity. When compared with chemotherapeutic regimens, pAd-T34A was as effective as taxol and considerably more effective than adriamycin in induction of tumor cell apoptosis and enhanced taxol-induced cell death. In three xenograft breast cancer models in immunodeficient mice, pAd-T34A suppressed de novo tumor formation, inhibited by approximately 40% the growth of established tumors, and reduced intraperitoneal tumor dissemination. Tumors injected with pAd-T34A exhibited loss of proliferating cells and massive apoptosis by in situ internucleosomal DNA fragmentation. These data suggest that adenoviral targeting of the survivin pathway may provide a novel approach for selective cancer gene therapy.

Interaction between the alpha-T catenin gene (VR22) and APOE in Alzheimer's disease.

BACKGROUND: APOE is the only gene that has been consistently replicated as a risk factor for late onset Alzheimer's disease. Several recent studies have identified linkage to chromosome 10 for both risk and age of onset, suggesting that this region harbours genes that influence the development of the disease. A recent study reported association between single nucleotide polymorphisms (SNPs) in the VR22 gene (CTNNA3) on chromosome 10 and plasma levels of Abeta42, an endophenotype related to Alzheimer's disease. OBJECTIVE: To assess whether polymorphisms in the VR22 gene are associated with Alzheimer's disease in a large sample of Alzheimer's disease families and an independent set of unrelated cases and controls. RESULTS: Several SNPs showed association in either the family based or case-control analyses (p<0.05). The most consistent findings were with SNP6, for which there was significant evidence of association in both the families and the unrelated cases and controls. Furthermore, there was evidence of significant interaction between APOE-4 and two of the VR22 SNPs, with the strongest evidence of association being concentrated in individuals carrying APOE-4. CONCLUSIONS: This study suggests that VR22 or a nearby gene influences susceptibility to Alzheimer's disease, and the effect is dependent on APOE status.

Expression of bone morphogenetic protein-4 (BMP-4), bone morphogenetic protein-7 (BMP-7), fibroblast growth factor-8 (FGF-8) and sonic hedgehog (SHH) during branchial arch development in the chick.

Expression of Fgf-8, Bmp-4, Bmp-7, and shh in the branchial arches of the chick embryo is examined by in situ hybridization. Fgf-8 expression is initially broad and diffuse, becoming more tightly restricted, particularly in the epithelium of the posterior ectodermal margin (PEM) of the 2nd branchial arch. Bmp-7 transcripts, first seen at stage 12 in discrete regions corresponding to the developing branchial clefts, are later detected in both clefts and arches, including the PEM of the 2nd arch while Bmp-4 transcripts are detected at stage 18 in the distal tips of the arches. Shh expression remains localized, overlapping with both Bmp-7 and Fgf-8 in the PEM of the 2nd arch at stages 16 and 18. Based on these data, a model is proposed for the role of these signalling molecules in branchial arch development.

Expression and modification of Hox 2.1 protein in mouse embryos.

A polyclonal antibody, alpha Hox 2.1a, has been generated and used to immunolocalize Hox 2.1 protein in mouse embryos. Protein is present in nuclei of all tissues previously shown to express Hox 2.1 RNA. In addition, protein is seen in somites and proximal regions of the limb buds, tissues in which Hox 2.1 RNA expression was not clearly detected previously by in situ hybridization. At the 7 somite stage, protein is detectable in the neural tube up to the level of somite 1, but later retracts to a more posterior position. Immunoblot, in vitro translation, and immunoprecipitation experiments were carried out to characterize the Hox 2.1 protein. The results show that the Hox 2.1 gene produces at least two related phosphorylated proteins present in different proportions in different tissues.

Frontal and temporal sources of mismatch negativity in healthy controls, patients at onset of schizophrenia in adolescence and others at 15 years after onset.

Mismatch negativity (MMN) is an event-related potential measure of auditory change detection. It is widely reported to be smaller in patients with schizophrenia and may not improve along with otherwise successful clinical treatment. The main aim of this report is to explore ways of measuring and presenting four features of frequency-deviant MMN dipole sources (dipole moment, peak latency, brain location and orientation) and to relate these to the processes of psychopathology and illness progression. Data from early onset patients (EOS) at the start of the illness in adolescence, and others who had their first break in adolescence 15 years ago (S-15Y) were compared with two groups of age-matched healthy controls (C-EOS, C-15Y). A four-source model fitted the MMN waveform recorded from all four groups, whether MMN amplitude was more (EOS) or less (S-15Y) reduced. The locations were in the left superior temporal and anterior cingulate gyri, right superior temporal and inferior/mid frontal cortices. Dipole latencies confirmed a bottom-up sequence of processing and dipole moments were larger in the temporal lobes and on the left. Patients showed small dipole location changes that were more marked in the S-15Y than the EOS group (more rostral for the left anterior cingulate, more caudal for the right mid-frontal dipole) consistent with illness progression. The modelling of MMN dipole sources on brain atlas and anatomical images suggests that there is a degree of dissociation during illness between small progressive anatomical changes and some functional recovery indexed by scalp recordings from patients with an onset in adolescence 15 years before compared to adolescents in their first episode.

The addition of bryostatin 1 to cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy improves response in a CHOP-resistant human diffuse large cell lymphoma xenograft model.

The incidence of non-Hodgkin's lymphoma has been increasing at a rate of 4% per year since 1950; more than 62,000 cases will be diagnosed in the United States in 2000. Diffuse large cell lymphoma (DLCL) is the prototype of curable non-Hodgkin's lymphoma. Empirically designed chemotherapy regimens did not increase the cure rate of 30-40% achieved by the original four-drug regimen introduced in the 1970s [cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP)]. We studied the antitumor effects of the CHOP regimen alone or in combination with a unique protein kinase C activator, bryostatin 1, on a xenograft model for resistant DLCL in mice with severe combined immune deficiency (WSU-DLCL2-SCID). In this model, the efficacy of bryostatin 1 given at 75 microg/kg, i.p., alone for 1 or 2 days [B(1x) and B(2x)]was compared with the efficacy of CHOP alone, bryostatin 1 + CHOP (B+CHOP) given concurrently, bryostatin 1 for 1 day followed by CHOP on day 2 [B(1x)-CHOP], and bryostatin 1 for 2 days followed by CHOP on day 3 [B(2x)-CHOP]. CHOP doses were as follows: (a) cyclophosphamide, 40 mg/kg, i.v.; (b) doxorubicin, 3.3 mg/kg, i.v.; (c) vincristine, 0.5 mg/kg, i.v.; and (d) prednisone, 0.2 mg/kg, every day for 5 days, p.o. Tumor growth inhibition (T/C), tumor growth delay (T-C), and log10 kill for B(1x), B(2x), CHOP, B+CHOP, B(1x)-CHOP and B(2x)-CHOP were 49%, 39%, 25.8%, 15.1%, 14.6%, and 12%; 6, 7, 16, 25, 12, and 15 days; and 0.6, 0.5, 2.2, 3.6, 1.7, and 2.0, respectively. To begin elucidating the mechanism whereby bryostatin 1 potentiated the effects of CHOP in the mouse model; we studied the effect of bryostatin 1 on Bax, Bcl-2, and poly(ADP-ribose) polymerase proteins in vitro and in vivo. Bax protein increased in a time-dependent manner without any measurable change in Bcl-2 expression. However, significant cleavage of the preapoptotic marker poly(ADP-ribose) polymerase was not recorded, and the percentage of apoptotic cells detected by flow cytometry increased only slightly (approximately 8%) after 96 h of bryostatin 1 exposure. The in vitro and in vivo results emphasize the superiority of combining bryostatin 1 with the CHOP regimen against the WSU-DLCL2 model. One possible mechanism may be the modulatory effects of bryostatin 1 on the Bax:Bcl-2 family of apoptosis-regulatory proteins. The use of this combination should be further explored clinically in the treatment of lymphoma.

Sequential treatment of human chronic lymphocytic leukemia with bryostatin 1 followed by 2-chlorodeoxyadenosine: preclinical studies.

We have previously reported that bryostation 1 (Bryo 1) induces differentiation of chronic lymphocytic leukemia (CLL) in vitro to a hairy cell (HC) stage. This study tests the hypothesis that Bryo 1-differentiated CLL cells are more susceptible to 2-chlorodeoxyadenosine (2-CdA) than parent CLL cells. A recently established EBV-negative CLL line (WSU-CLL) from a patient resistant to chemotherapy including fludarabine was used to test this hypothesis. Both Bryo 1 (10-1000 nM) and 2-CdA (5.6-22.4 microM) exhibited a dose-dependent growth inhibitory effect on the WSU-CLL cell line. In vitro, the sequential exposure to Bryo 1 (100 nM for 72 h) followed by 2-CdA (11.2 microM) resulted in significantly higher rates of growth inhibition than either agent alone. Changes in immunophenotype, enzymes, lipids, proteins, and the DNA of WSU-CLL cells were studied before and after Bryo 1 treatment. Bryo 1 induced a positive tartrate-resistant acid phosphatase reaction and two important markers, CD11c and CD25, after 72 h of culture, confirming the differentiation of CLL to HC. The Fourier transformation infrared spectroscopic analysis showed that the amount of membrane lipids significantly increased in Bryo 1-treated cells compared to controls after 24 h, whereas the protein content, as well as the DNA content, decreased. This finding supports the change of CLL to HC. To evaluate the in vivo efficacy of Bryo 1 and 2-CdA, we used a xenograft model of CLL in WSU-CLL-bearing mice with severe combined immune deficiency. s.c. tumors were developed by injection of 10(7) WSU-CLL cells, and fragments were then transplanted into a new batch of severe combined immunodeficient mice. Bryo 1 and 2-CdA at the maximum tolerated doses (75 micrograms/kg i.p. and 30 mg/kg s.c., respectively) were administered to the mice at different combinations and schedules. The survival in days, the tumor growth inhibition ratio, the tumor growth delay, and the log10 kill of the mice treated with Bryo 1 followed by 2-CdA were significantly better than the control and other groups. We conclude that the sequential treatment with Bryo 1 followed by 2-CdA resulted in higher antitumor activity and improved animal survival.

Phase II trial of bryostatin 1 in patients with relapsed low-grade non-Hodgkin's lymphoma and chronic lymphocytic leukemia.

Bryostatin 1 is a natural product isolated from the marine bryozoan Bugula neritina in 1982 and is currently undergoing evaluation in a number of malignancies. Twenty-five patients with relapsed, low-grade non-Hodgkin's lymphoma or chronic lyphocytic leukemia (CLL) received bryostatin 1 by 72-h continuous infusion every 2 weeks at a dose of 120 microg/m2 per course. Patients who progressed while receiving bryostatin 1 alone could participate in a feasibility study by receiving vincristine administered by bolus i.v. injection immediately after the completion of the bryostatin 1 infusion. The dose of vincristine was escalated in groups of three patients as follows: level 1, 0.5 mg/m2; level 2, 1.0 mg/m2; and level 3, 1.4 mg/m2 with vincristine doses capped at 2.0 mg for all patients. Bryostatin 1 alone resulted in one complete remission and two partial remissions. Nine patients received sequential treatment with bryostatin 1 and vincristine. The addition of vincristine at a dose of 2 mg was feasible and caused the expected dose-related sensory neuropathy. Phenotypic analysis by flow cytometric analysis on pre- and post-bryostatin 1-treated peripheral blood lymphocytes revealed up-regulation in the coexpression of CD11c/ CD22 on CD20+ B cells in two of four CLL patients studied, which is consistent with in vitro findings of differentiation of CLL cells to a hairy cell phenotype.

Pituitary thyrotropin (TSH) rebound phenomenon and kinetics of secretion in the goitrous rat: differential effects of thyroxine on synthesis and release of TSH.

The protein metabolism and [3H]-uridine uptake of thyroid and adenohypophysis and the kinetics of pituitary TSH rebound (PTR) were studied in goitrous female rats (fed propylthiouracil, PTU: for 7-12 weeks) following single, iv injections of L-thyroxine (T4: 0.8 to 200 mug). Goitrogenesis was associated with reduced protein concentration and enhanced uptake of [3H] uridine in both glands. Plasma levels of TSH were invariably elevated but stores in the adenohypophysis were consistently reduced. Small doses of T4 (4 mug) induced significant TSH repletion in the pituitary within 2-6 h following injection. Accumulations of pituitary TSH to supranormal levels (15-fold increases) were achieved with 20 mug T4 at 6 and 24 h; higher doses (100-200 mug) inhibited the PTR at all time intervals tested (0.5-24 h). Administration of puromycin or actinomycin D did not influence the PTR. Protein content and labeled uridine uptake of the pituitary bore no apparent relationship to T4-induced TSH repletion in the gland. Blood clearance rate of exogenous rat TSH was measured prior to and during PTR. Plasma half-life was determined to be 13.6 and 19.9 min in euthyroid and chronically hypothyroid rats, respectively; it was not significantly altered from the latter during rebound (18.7 min). Calculations of theoretical TSH secretory rates prior to (50.5 +/- 4.4 mU/h) and after rebound with 20 mug T4 (25.4 +/- 4.2 mU/H) revealed that the reaccumulation of TSH in the pituitary induced with T4 cannot be attributed solely to inhibition of release, but may also involve enhancement of synthesis. It is concluded that T4 administration at high dose levels inhibits both synthesis and release of TSH from pituitary thyrotrophs, whereas low critical doses of T4 suppress release, but augment synthesis and/or facilitate conformational change in a pituitary precursor(s) molecule which renders it detectable by bioassay.

Bax:Bcl-2 ratio modulation by bryostatin 1 and novel antitubulin agents is important for susceptibility to drug induced apoptosis in the human early pre-B acute lymphoblastic leukemia cell line, Reh.

The ratio of Bax to Bcl-2 protein can determine whether cells will die via apoptosis or be protected from it. Reh was found to express a high basal level of Bcl-2 but was lacking of Bax protein expression. Treatment with bryostatin 1 induced a down-regulation in Bcl-2 protein that was not accompanied by an obvious Bax protein induction or apoptosis. These results suggest that a decreased level of Bcl-2 alone in this cell line is not sufficient for apoptosis induction. In an effort to identify the mechanism whereby apoptosis could be induced in this ALL model, we treated Reh cells with three microtubule inhibitors: dolastatin 10, auristatin PE and vincristine, in the presence and absence of bryostatin 1. When used alone, only dolastatin 10 induced apoptosis that was detected morphologically, and by flow cytometry. Western blots revealed that dolastatin 10-induced apoptosis was accompanied by the induction of Bax protein and the reduction in Bcl-2 protein. Auristatin PE and vincristine induced both Bax and Bcl-2 protein, leaving the Bax:Bcl-2 ratio constant. Reh cells pretreated for 24 h with bryostatin 1 followed by dolastatin 10, auristatin PE or vincristine showed significant apoptosis which was accompanied by Bcl-2 protein down regulation and Bax protein up regulation. We conclude that: (1) expression of bax is necessary for apoptosis-induction in this model; (2) a decrease in Bcl-2 level alone is not sufficient and might not be necessary for apoptosis-induction; and (3) the ratio of Bax:Bcl-2 plays a critical role in susceptibility to apoptosis in Reh cells. The results from this study should prove useful in guiding the clinical application of these novel agents in the treatment of acute lymphoblastic leukemia.

Treatment of a de novo fludarabine resistant-CLL xenograft model with bryostatin 1 followed by fludarabine.

WSU-CLL is a de novo fludarabine resistant cell line established from a patient with advanced chronic lymphocytic leukemia (CLL) refractory to chemotherapy including fludarabine (Flud). Our previous studies indicate that bryostatin 1 (Bryo 1) induces differentiation of WSU-CLL and increases the ratio of dCK/5'-NT activity and Bax/Bcl-2. This study tests the hypothesis that Bryo 1-differentiated cells are more susceptible to Flud than the parent WSU-CLL cells. Flud, given sequentially after Bryo 1, in vitro and in vivo animal studies resulted in significantly higher rates of growth inhibition and improved animal survival. Flud at 100 to 600 nM exhibited a dose-dependent growth inhibitory effect on the WSU-CLL cell line. The sequential exposure to Bryo 1 (10 nM for 72 h) followed by Flud (100 nM) resulted in significantly higher rates of growth inhibition than either the reverse addition of these two agents or each agent alone, but was not significantly different than the concurrent addition of Bryo 1 + Flud. Using 7-amino-actinomycin D staining and flow cytometry, apoptosis was seen in 40.8% of cells treated with Bryo 1 (10 nM, 72 h) followed by Flud, compared with Flud (100 nM, 72 h) followed by Bryo 1 (18.1%). To demonstrate that Bryo 1 enhancement of Flud efficacy was not restricted to in vitro culture, we used the WSU-CLL xenograft model in mice with severe combined immune deficiency (SCID). Bryo 1 + Flud at the maximum tolerated doses (75 microg/kg i.p. and 200 mg/kg i.v., respectively) were administered to mice in different combinations. The survival in days, the tumor growth inhibition ratio (T/C), the tumor growth delay (T-C) in days, log10 kill, as well as mean tumor weight (mtw) of mice treated with Bryo 1 followed by Flud, were significantly better than control and other groups. T/C%, T-C, log10 kill and mtw were as follows: Bryo 1 (36.8%, 10 days, 0.8, 375 mg); Flud (100%, 0. 0 day, 0.0, 1130 mg); Bryo 1 + Flud (14.3%, 12 days, 0.95, 288 mg); Bryo 1 followed by Flud (4.6%, 17 days, 1.35, 35 mg); Flud followed by Bryo (40.3%, 10 days, 0.80, 175 mg). We conclude that: i) Bryo 1 sensitizes WSU-CLL cells to Flud and enhances apoptosis; ii) the sequential treatment with Bryo 1 followed by Flud resulted in higher anti-tumor activity compared with either agent alone, in combination, or the reverse addition of these agents and iii) these results are comparable to those of Bryo 1 followed by 2-CdA suggesting common pathway(s) of interaction between Bryo 1 and purine analogues.