Eileen Bryant

Negative regulators of hemopoiesis and stroma function in patients with myelodysplastic syndrome.

The mechanism that leads to hemopoietic failure in patients with myelodysplastic syndrome (MDS) is not well understood. There is evidence, however, that regulatory molecules such as tumor necrosis factor (TNF)-α, Fas (CD95), and Fas-ligand, which negatively affect hemopoiesis by way of apoptosis are upregulated. Here we analyzed marrow samples from 80 patients with MDS in regard to TNF-α and Fas-ligand levels and a possible correlation with various disease parameters and risk factors. TNF-α levels were elevated in comparison to samples from normal marrow donors, however, no significant correlation with FAB subtype, cytogenetic risk group or score by the International Prognostic Scoring System (IPSS) was observed. However, there was an inverse correlation between the cytogenetic risk category (low, intermediate, high) and levels of soluble Fas-ligand. The major source of TNF-α were mononuclear (non-stromal) cells which appeared to produce TNF-α at maximum levels. Limiting dilution analysis of CD34+ precursor cells showed that individually assayed cells, removed from companion cells that presumably provided negative signals such as TNF-α or Fas-ligand, were able to generate progressively increasing numbers of colonies. Stromal layers derived from MDS marrow did not have an inhibitory effect. In fact, higher colony numbers were obtained from both normal and MDS marrow derived hemopoietic precursors propagated on irradiated stromal layers from MDS marrow than on stromal layers from normal marrow. These results show that substantial numbers of normal hemopoietic precursors persist in MDS marrow. However, differentiation into mature cells is inhibited by negative signals originating from accessory or abnormal hemopoietic precursors in the non-adherent marrow fraction.

Cultivated cells from diagnostic amniocentesis in second trimester pregnancies. I. Clonal morphology and growth potential.

Extract: Amniotic fluid obtained transabdominally for prenatal diagnosis (mean length of amenorrhea 16.1 weeks) contained between 10 and 10 cells/ml, the great majority of which were squamous; their labeling index with ( H)thymidine was < 2 × 10-3. An average of 3.5 clones ( > 10 cells)/ml fluid grew; of these, an average of 1.5 grew for >20 population doublings (CPD). Of 288 clones analyzed from 20 cases, 271 could be classified into one of three groups: group I, 24 were typical fibroblast-like cells (F cells) comparable with control clones from neonatal dermis with macroscopic “ribbing” growth pattern of megaclones, preponderance of spindle-shaped cells growing in parallel arrays, and high growth potential (mean of selected clones = 53 CPD); group II, 67 were typical epithelioid types (E cells) consisting of cells with intimate cell-to-cell contact which were resistant to trypsin detachment, with poor subcloning efficiencies and poor growth potential (mean of selected clones 14.5 CPD); group III, the predominant class with 180 clones, was of a type not described previously (AF cells); their megaclones had “bulls-eye” growth patterns, individual cells were pleomorphic, and growth potentials were intermediate to those of E and F cells. This tripartite classification holds for the majority of samples, but individual variation is suggested by the observation of exceptional clonal types sharing characteristics of both the E and AF class.Speculation: The AF cell is the predominant type in most diagnostic amniotic fluid cultures; heretofore, such cultures were widely regarded as consisting primarily of fibroblast and epithelial-like cells. Skin fibroblast cultures are therefore inappropriate controls for the interpretation of constitutive and induced levels of enzymes in such cultures. Established amniotic fluid cell cultures are derived from a single or a few clonable cells with high growth potential and therefore may not always be representative of the fetus.

Failure of adult marrow-derived stem cells to generate marrow stroma after successful hematopoietic stem cell transplantation

OBJECTIVE The existence of adult, marrow-derived stem cells that retain the ability to generate various tissues is an appealing concept that has considerable therapeutic potential. The aim of this study was to test the extent of this proposed plasticity by defining the ability of adult marrow and peripheral blood stem cells to generate stromal cells of the marrow microenvironment. PATIENTS AND METHODS We examined expanded populations of stromal cells from four patients 1 to 27 years after allogeneic, sex-mismatched marrow, or peripheral blood stem cell transplantation. The cultured stromal cells were stained by immunofluorescence and with nonspecific esterase (NSE) to detect macrophages, which can constitute a significant component of a primary long-term marrow culture. Fluorescence in situ hybridization (FISH) probes for chromosomes X and Y were applied to distinguish donor from host cells. RESULTS FISH analysis of replicate slides indicated a good correlation between the number of NSE(+) cells and the number of donor-derived cells. By applying NSE and FISH to the same cells and capturing both bright-field and epifluorescence images, we confirmed that all donor signals were derived from NSE(+) macrophages. CONCLUSION After successful allogeneic stem cell transplantation, the marrow stroma remains host in origin, even after 27 years of 100% donor hematopoiesis.

p53 and p21 Form an Inducible Barrier that Protects Cells against Cyclin E-cdk2 Deregulation

BACKGROUND Cyclin E, in conjunction with its catalytic partner cdk2, is rate limiting for entry into the S phase of the cell cycle. Cancer cells frequently contain mutations within the cyclin D-Retinoblastoma protein pathway that lead to inappropriate cyclin E-cdk2 activation. Although deregulated cyclin E-cdk2 activity is believed to directly contribute to the neoplastic progression of these cancers, the mechanism of cyclin E-induced neoplasia is unknown. RESULTS We studied the consequences of deregulated cyclin E expression in primary cells and found that cyclin E initiated a p53-dependent response that prevented excess cdk2 activity by inducing expression of the p21Cip1 cdk inhibitor. The increased p53 activity was not associated with increased expression of the p14ARF tumor suppressor. Instead, cyclin E led to increased p53 serine15 phosphorylation that was sensitive to inhibitors of the ATM/ATR family. When either p53 or p21cip1 was rendered nonfunctional, then the excess cyclin E became catalytically active and caused defects in S phase progression, increased ploidy, and genetic instability. CONCLUSIONS We conclude that p53 and p21 form an inducible barrier that protects cells against the deleterious consequences of cyclin E-cdk2 deregulation. A response that restrains cyclin E deregulation is likely to be a general protective mechanism against neoplastic transformation. Loss of this response may thus be required before deregulated cyclin E can become fully oncogenic in cancer cells. Furthermore, the combination of excess cyclin E and p53 loss may be particularly genotoxic, because cells cannot appropriately respond to the cell cycle anomalies caused by excess cyclin E-cdk2 activity.

USE OF (CA)N POLYMORPHISMS TO DETERMINE THE ORIGIN OF BLOOD CELLS AFTER ALLOGENEIC CANINE MARROW GRAFTING

We have used a polymerase chain reaction-based assay measuring polymorphic (CA)n repeats, a class of simple sequence repeats, to assess the success of allogeneic canine marrow transplants. Results were compared with those obtained with karyotype analysis of dividing cells in recipients that were sex mismatched with their marrow donors. Twenty recipients were conditioned for transplantation of genotypically DLA-identical littermate marrow by 450 cGy of total-body irradiation. In 2 recipients, results could not be compared, since either only cytogenetic or dinucleotide (CA)n marker data existed. Both dogs had autologous marrow recovery. In 15 of the remaining 18 recipients, complete agreement was found between the results obtained with dinucleotide (CA)n markers, cytogenetic studies, and granulocyte changes after transplantation. Seven of the 15 showed eventual autologous recovery, 6 displayed mixtures of host and donor cells, and 2 showed donor-type hematopoiesis. Two of the 18 dogs showed mixed chimerism with (CA)n markers and autologous recovery by cytogenetics, findings that may be related to differences in cells analyzed by the two techniques–i.e., all nucleated cells by (CA)n markers versus dividing cells by cytogenetics. In one additional recipient, results of marrow cytogenetics, granulocyte changes, and (CA)n markers were consistent with a successful allograft, while peripheral blood cytogenetics suggested autologous recovery, possibly the result of erroneous blood sampling. Polymerase chain reaction-based testing for dinucleotide repeat (CA)n polymorphisms, originally developed for genetic mapping in the dog, is useful and reliable when compared with cytogenetic studies, in assessing the success of allogeneic marrow transplants in dogs.

Systematic growth studies, cocultivation, and cell hybridization studies of Werner syndrome cultured skin fibroblasts

SummaryThe growth of 20 independently derived skin fibroblastlike (FL) cell strains from three individuals with Werner syndrome (adult progeria) was compared with the growth of ten FL cell strains from normal individuals. Population growth rates and total replicative life spans of Werner syndrome strains averaged 55% and 27%, respectively, of the growth rates and life spans of non-Werner strains. In the first few passages, four Werner syndrome strains demonstrated population growth rates in the low normal range, but the longest-lived Werner syndrome strain had only 75% of the total replicative potential of the shortest-lived normal strain. Exponential growth rates, cloning efficiencies, and saturation densities of Werner strains were also reduced, whereas cell attachment was normal. Viable cells (identified by dye exclusion) were maintained in post-replicative Werner syndrome and control cultures for periods of at least 10 months; there was no evidence of accelerated post-replicative senescence or cell death of Werner syndrome FL cells. Cocultivation of Werner syndrome and normal strains did not influence population growth rates of either strain. Two proliferating hybrid clones were obtained from fusions of normal and Werner syndrome FL cell strains and these hybrids displayed the reduced growth potential typical of Werner syndrome FL cells. These studies confirm that low growth rate and sharply reduced replicative life span are characteristic of cultured skin FL cells from patients with Werner syndrome, and they suggest that these characteristics are not affected by complementation with non-Werner FL cells.

Predictors of relapse and overall survival in Philadelphia chromosome-positive acute lymphoblastic leukemia after transplantation.

Allogeneic transplantation offers a potential cure for patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). We performed a retrospective analysis examining pretransplantation and posttransplantation prognostic factors in 90 patients with Ph+ ALL. The median age of the patients was 33 years, with slightly more than half of the patients (58%) in clinical remission at the time of transplantation. Overall, patients had a nonrelapse mortality rate of 30%, a relapse percentage of 34%, and an estimated 5-year disease-free survival rate of 30%. Pretransplantation risk factors for relapse included the expression of the p190 transcript (relative risk [RR] = 5.1; P =.037), evidence of morphologic disease at the time of transplantation (RR = 3.9; P <.001), and type of donor (RR = 2.5; P =.015), with patients receiving autologous or matched related transplants having the highest risk of relapse. The detection of minimal residual disease by reverse transcription polymerase chain reaction for bcr-abl transcripts was a significant posttransplantation risk factor for relapse (RR = 4.4; P =.001), with posttransplantation patients expressing the p190 transcript having the highest risk of relapse (RR = 8.7; P =.0001). In addition, patients with chronic extensive graft-versus-host disease showed a significantly lower risk of relapse (RR = 0.33; P =.038). Thus, these findings indicate that several pretransplantation and posttransplantation risk factors exist for patients with Ph+ ALL. Together, these factors can be used to improve our risk stratification of patients with Ph+ ALL who undergo transplantation, which will greatly enhance our ability to counsel these patients and potentially lead to the development of more specific treatment plans for them.

Association of an apolipoprotein CII allele with familial dementia of the Alzheimer type

In order to identify the genetic locus responsible for familial dementia of the Alzheimer type (DAT), we are studying 10 families in which DAT appears to be inherited as an autosomal dominant trait. Genotypes for a TaqI restriction fragment length polymorphism (RFLP) at the apolipoprotein CII locus were determined for the following groups: affected and unaffected DAT family members, DAT subjects with no family history of the disease, and normal control subjects. The control group included 103 individuals from our study and 123 from the study of Wallis et al. (Hum. Genet., 68 (1984) 286). The frequency of the TaqI fast (F) allele in the affected familial DAT subjects (0.64 +/- 0.08) differed significantly from that for the control group (0.39 +/- 0.02) (Z = 2.87, P less than 0.005). In contrast, the F-allele frequency for the unaffected family members was 0.31 +/- 0.09, which was similar to that of the combined control group (Z = 0.78, P greater than 0.40). Subsequently, genotypes were determined for two other polymorphisms at the Apo CII locus: a BanI RFLP and a BglI RFLP. For these two polymorphisms, the allele frequencies for the familial DAT subjects differed from the unaffected control groups but the differences were smaller and not statistically significant. These data suggest a previously unrecognized association between the Apo CII TaqI F-allele and familial DAT.

Growth Characteristics of Werner Syndrome Cells in Vitro

Cultured skin fibroblast-like (FL) cells from patients with Werner syndrome grow poorly: reduced growth potential has been reported by more than 11 laboratories for cultures from more than 26 patients, using many different tissue culture media. This characteristic is of importance to research in Werner syndrome for several reasons. It remains to be determined whether poor growth is a primary or a secondary manifestation of the basic molecular defect, and the relationship is not yet clear between reduced growth of cultured skin fibroblasts and the other manifestations of Werner syndrome in vitro and in vivo. This characteristic also makes it difficult to obtain sufficient growth for clonal studies or for biochemical studies that require a large number of cells.

Marrow transplantation for Fanconi anaemia: conditioning with reduced doses of cyclophosphamide without radiation.

Nine patients with Fanconi anaemia (FA) were conditioned for HLA‐identical sibling bone marrow transplant (BMT) with reduced dose of cyclophosphamide (Cy) without radiation or antithymocyte globulin (ATG). The total dose of Cy was 140 mg/kg (n=2) or 120 mg/kg (n=7). The median patient age was 8 years (range 4–19). Graft‐versus‐host disease (GVHD) prophylaxis was with methotrexate and cyclosporine (n=8) or cyclosporine alone (n=1). All patients had sustained engraftment and two developed grade ≥II acute GVHD. Cy toxicity included grade ≥2 mucositis seen in all evaluable patients and haemorrhagic cystitis in two patients. The Kaplan‐Meier survival estimate is 89% with a median follow‐up of 285 d (range 56–528).