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Fanconi's anaemia is a rare genetic disorder and majority of the patients die of haematologic complications in their second or third decades of life. Others who have mild or no cytopenias survive long enough to develop malignancies. This is a report of a 44-year-old woman who presented with recurrent oral squamous cell carcinoma during her adulthood, without clinical haematological problem. Despite treatment with cis-retinoic acid, she developed a third squamous cell carcinoma 6 months later. In a review of the literature, only in 1 reported case was the patient treated with low-dose retinoids but he developed recurrent anal cancer after 14 months.

The polypeptide encoded by the Fanconi anemia (FA) complementation group C gene, FAC, binds to a group of cytoplasmic proteins in vitro and may form a multimeric complex. A known mutant allele of FAC resulting from the substitution of Pro for Leu at codon 554 fails to correct the sensitivity of FA group C cells to mitomycin C. We reasoned that overexpression of the mutant protein in a wild-type cellular background might induce the FA phenotype by competing with endogenous FAC for binding to the accessory proteins. After stable transfection of 293 cells with wild-type and a mutant FAC allele containing the L554P substitution, four independent clones that expressed four-to-fifteen fold higher levels of transcript from the mutant transgene relative to the endogenous FAC gene showed hypersensitivity to mitomycin C. By contrast, both parental and FAC-overexpressing cells maintained their relative resistance to mitomycin C. No differences in the biosynthesis, subcellular localization and protein interactions of the normal and mutant proteins were detected. The induction of the FA phenotype in this system is compatible with the competition hypothesis and provides support for a functional role of the FAC-binding proteins in vivo.

Bone marrow failure in the pediatric patient places the hematologist at the junction of clinical medicine, cellular biology, and molecular genetics. The pathophysiology of these disorders is rapidly being elucidated in many laboratories. Treatments such as bone marrow transplantation and the nascent modality of gene therapy are firmly grounded in these modern sciences. This year's progress in the understanding, diagnosis, and treatment of a wide variety of predominantly pediatric bone marrow failure states is a direct result of the union between the "laboratory bench and the patient bedside."

The potential of enhanced chromatid damage, observed after X-irradiation of G2 phase, has been used to detect individuals genetically predisposed to cancer, utilising fibroblast/lymphocytes from these patients as well as fibroblasts derived from human tumours. Fibroblasts and/or lymphocyte samples of two autosomal recessive syndromes (xeroderma pigmentosum (XP), Fanconi's anaemia (FA)) and one congenital or acquired disorder, aplastic anaemia (AA), were employed for the G2 radiosensitivity assay. In addition, we have estimated the frequencies of spontaneously occurring chromosomal aberrations as well as G2 radiosensitivity of eight samples of fibroblasts/fibroblast-like cells (two normal, two colorectal carcinoma, two Wilms' tumour, one retinoblastoma and one polyposis coli), and three samples of lymphocytes (two normal and one from a lymphoma patient). The results obtained indicate that there were no differences between fibroblast cells derived from patients or tumours, except FA patients, in the frequency of spontaneously occurring chromosomal aberrations when compared to normal cells. Following X-irradiation we did not observe any significantly increased G2 radiosensitivity in FA and XP cells. Lymphocytes from AA and lymphoma patients, and all tumour cell lines except retinoblastoma, responded with increased frequencies of aberrations following G2 X-irradiation in comparison to cells derived from normal individuals. In our hands, the G2 sensitivity assay could not always discriminate cells from cancer-prone individuals from those of controls.

Morphologically, bone marrow is made up of a relatively mature but heterogenous population, fueled by a tiny pool of microscopically unrecognizable stem and progenitor cells. This complex tissue has the responsibility of maintaining our hematopoietic and, to a large extent, immunologic integrity, both of which are indispensable for health and, indeed, survival. Perhaps not surprisingly, bone marrow is the target of genetic, autoimmune, and environmental insults. Although robust, it has only a limited number of responses, one of which is reduction in cellular output, sometimes with superimposed qualitative abnormalities, and this is defined as bone marrow failure. Bone marrow failure is a diverse entity but can be logically explained and classified on a pathophysiologic basis. Thus the major recognizable categories of bone marrow failure are congenital and acquired defects. Each of these is subdivided according to the number of cell lines involved, over and above which the severity of the damage will determine reversibility. In each case, the natural history dictates management, and this ranges from short-term growth factor support to biologic immune response modulation and finally to bone marrow transplantation. In the past, many clinicopathologic variants of bone marrow failure were described, although their etiology was obscure and effective therapy was unavailable. This changed dramatically, however, when experimental hematologists, using radiobiology models, uncovered the dynamic nature of blood formation. Cardinal observations included the way in which spontaneous recovery followed irradiation, the central role played by pluripotential stem cells, and the integral participation of stroma in modulating this entire process. Understanding was refined once bone marrow cultures became available while, in parallel, the use of in-bred mouse strains launched the era of allogeneic transplantation. These approaches were combined, and the broad principles that govern basal or constitutive production emerged. Stem cells, with their characteristic commitment to self-renewal, exist at the apex of a hierarchy and generate a tier of proliferating progenitors that, in turn, give rise to a large postmitotic compartment of precursors that mature into distinctive myeloid and lymphoid lineages. The reserve potential is enormous, and output can be induced to meet even greatly increased demands. These events reflect the interaction of growth factors with a balancing set of negative regulators. The link between such diverse functions resides, to a large extent, in accessory cells and matrix geographically organized in what is now described as the hematopoietic inductive microenvironment. Many details of these meticulously orchestrated processes are obscure.(ABSTRACT TRUNCATED AT 400 WORDS)

Culture of bone marrow from patients with Diamond-Blackfan anaemia (DBA) has previously shown a variable progenitor response to growth factor stimulation. An extensive standardized study has now been undertaken to investigate the presence of distinct sub-groups in this disorder. In vitro response of bone marrow progenitors to recombinant human growth factors, including stem cell factor, was examined in 18 DBA patients and five normal donors, assessing BFU-E, CFU-GM and CFU-GEMM development. In 16 of the DBA patients a synergistic response to combinations of growth factors was observed with optimal growth in cultures containing erythropoietin, interleukin-3 and stem cell factor. Growth factor induced erythroid response formed three distinct groups, based on BFU-E numbers: type I (mean age 4.87 years) showed > 70% normal erythroid response; type II (mean age 13.87 years) showed < 70% normal; and type III (mean age 15.29 years) < 5% normal. CFU-GM response also followed the trigrouping. The results suggest more than one pathogenic mechanism for the erythroid failure in DBA, indicating DBA may be composed of more than one distinct disorder, and further suggest the defect in DBA may not be confined to the erythroid series.

The effect of caffeine on the endogenous G2 phase cell cycle blockage of Fanconi anemia (FA) cells was compared with the effect of caffeine on the G2 phase blockage induced in control cells by X-irradiation. The G2 phase accumulations in FA cells could be completely resolved by exposure to 1.5 mM caffeine. This was also observed in three brothers with endogenous G2 phase blockage due to unusual BrdU sensitivity. In contrast, G2 phase blockage induced by X-irradiation was only partially resolved by exposure to caffeine. The rescued G2 phase cells from FA patients were arrested within the following G1 phase compartments. This was not seen in X-irradiated cells from control donors. These results point towards a different nature and/or repair mechanism of the endogenous G2 phase lesion in FA cells compared to that induced by X-irradiation in control cells.

Mechanisms involved in the erythroid failure characterizing Diamond-Blackfan anaemia (DBA) remain unidentified. The general consensus is that the defect is intrinsic to the marrow erythroid progenitor, but the target progenitor cell has not been precisely identified, and in vitro studies have revealed considerable heterogeneity between patients. In order to understand better the meaning of such a biological heterogeneity, we examined the in vitro response of erythroid progenitors CFU-E (colony-forming unit-erythroid) and BFU-E (burst-forming unit-erythroid) to erythropoietin (Epo), interleukin-3 (IL-3) and stem cell factor (SCF) in a large series of 24 patients from 1 month to over 20 years of age. Results of colony assays revealed a striking correlation between the age of the patient and the extent of the abnormalities detected in vitro. Therefore, despite profound anaemia, 80% (7/10) of the patients studied within 1 year of diagnosis had normal numbers of both CFU-E and BFU-E which exhibited a normal response to cytokines. In contrast, 12/14 patients followed up for more than 3 years had decreased numbers of erythroid progenitors, in seven cases associated with decreased colony-forming unit granulocyte-macrophage (CFU-GM). The number of CFU-E and BFU-E was not normalized even by the addition of high concentrations of combined Epo, IL-3 and SCF.(ABSTRACT TRUNCATED AT 250 WORDS)

In two previous studies, we observed that recombinant human interleukin-3 (IL-3) induced an increase in marrow burst-forming unit-erythroid-derived colonies in vitro in some patients with Diamond-Blackfan anemia (DBA). To determine whether a similar erythropoietic response could be induced in vivo, we treated 13 patients with DBA (aged 4 to 19 years) with two preparations of IL-3. All patients had absent absolute reticulocyte counts and markedly reduced to absent recognizable bone marrow erythroid elements; patients with circulating reticulocytes in the previous 12 months were excluded from study. All patients except 1 had failed steroid therapy and had been transfusion-dependent since infancy; 1 patient was maintained on high-dose prednisone at the time of enrollment. On the first arm of the study, IL-3 (Immunex Corp, Seattle, WA) was administered subcutaneously using a dose escalation regimen of 125 to 500 micrograms/m2/day in divided dosage at 12-hour intervals, coadministered with 1.5 mg/kg/d of oral ferrous sulphate. Of the 13 patients that entered the trial, 4 stopped prematurely because of adverse side effects. In the other 9 evaluable cases, reticulocytes increased transiently in 1 patient from 0 to 65 x 10(9)/L after 35 days of IL-3 therapy at 250 micrograms/m2, but transfusion dependency persisted. One transient peak in absolute reticulocyte count was noted in 6 other patients, but no erythroid response was observed after completion of a full course of IL-3. Oral prednisone at 0.5 mg/kg/d was then coadministered with IL-3 at 500 micrograms/m2 to 5 of the patients without effect, and treatment was stopped. In 2 patients, a second preparation of IL-3 (Sandoz Canada Inc, Dorval, Quebec, Canada) was initiated in a dose escalation regimen of 2.5 to 10 micrograms/kg and was coadministered with ferrous sulphate. No erythroid response was observed in either patient, and in one of the two, alternate-day subcutaneous recombinant erythropoietin at 300 U/kg was administered for 3 weeks in combination with daily IL-3 at 10 micrograms/kg, but no increased erythropoiesis was seen. Significant increases in white blood cell and eosinophil counts during administration of both preparations of IL-3 were observed in all patients. These data show that the response of DBA patients to IL-3 in vivo is heterogeneous and cannot be predicted from in vitro studies. The absence of a corrective effect of IL-3 in these patients with DBA indicates that a deficiency of the cytokine is not central in the pathogenesis of the disorder.

This review summarizes both historical and more recent data on the clinical, cellular and genetic features of Fanconi anemia (FA), a rare autosomal recessive disorder. FA patients are characterized by pancytopenia, congenital malformations, growth delay and an increased susceptibility to the development of malignancies, particularly acute myelogenous leukemia. FA cells show chromosomal fragility, slow growth and increased sensitivity to DNA crosslinking agents. FA can be caused by defects in any one of at least four genes. Two general hypotheses have been proposed to explain the underlying defect: loss of a DNA repair function or of a step in the defense toward oxygen toxicity. After many attempts to clone the FA genes, the first one, that defective in group C, has been cloned by complementation of the increased sensitivity of FA(C) cells to mitomycin C and diepoxybutane. This gene (FACC) codes for a novel protein and is ubiquitously expressed. Mutations in various FA(C) patients that cause loss of function have been identified. The review concludes by suggesting directions for future research in FA.

Prenatal diagnosis in the third pregnancy of a mother who already had one healthy son and one son with Fanconi anaemia (FA), revealed that her fetus was also affected with FA. At 22 weeks a maternal complaint about excessive fetal kicking starting at 15 weeks, focused our attention on the behaviour of the fetus, which was observed by means of real-time ultrasound for 30 min. The differentiation of specific movement patterns was strongly diminished. The qualitative expression of general movements was considered to be consistently abnormal due to the fact that they were performed with large amplitudes, high speed and abrupt onsets. The incidence of general movements was within the normal range, however, the distribution in the burst-pause pattern was abnormal. Postmortem examination showed a spongy myelinopathy of the central nervous system that may account for the abnormal motor activity. This combination of findings has not been previously reported in association with FA.

Fanconi anemia is an autosomal recessive disease characterized by a high risk of developing bone marrow (BM) failure and acute myelogenous leukemia. We studied growth of hematopoietic progenitor cells in long-term BM culture (LTBMC) in 8 persons with Fanconi anemia and BM failure. Although LTBMC were initiated with very few BM cells, an adherent layer formed in cultures from 7 persons. In these cultures, the number of nonadherent cells increased for 10 to 15 days. Cell growth continued until cultures were terminated at day 35 to 40. During the first 2 weeks of culture, most nonadherent cells were differentiated myeloid cells. By days 35 to 40, the adherent layer contained cells able to initiate secondary LTBMCs. These data indicate that hematopoietic precursors cells able to proliferate and differentiate in vitro are present in the BM of persons with Fanconi anemia and BM failure. They suggest that mechanisms other than absent precursor cells are responsible for BM failure in Fanconi anemia.

Diamond-Blackfan anemia (DBA) is pleiotropic clinically and in vitro, and there is a strong suspicion that DBA is really a family of diseases that shares a common hematological phenotype. Although standard clonogenic assays of erythroid progenitors have been very informative about pathogenesis, they are not diagnostic of DBA, have no relationship to the clinical presentation and do not relate to the hemoglobin level or to the percentage of marrow erythroids at the time of study. Studies on progenitor-enriched marrow cells have furthered our understanding of DBA and have clearly shown marked differences among patients with respect to erythropoietin and "burst-promoting activity" responsiveness. In vitro addition of corticosteroids, interleukin 3 (IL-3) and/or Steel factor has produced a corrective effect on erythropoiesis in some DBA patients and has prompted clinical trials with IL-3 with variable results. It is clear that there is a disparity between the vitro data and clinical outcome, and therefore, the erythroid progenitor responsiveness to steroids and cytokines has limited predictive value clinically. Based on more than two decades of study, a model of DBA has evolved based on putative blocks at various stages along the erythropoietic differentiation pathway. These blocks likely represent a disorder of receptor-ligand interaction involving one or more growth-promoting cytokines.

Diamond-Blackfan anaemia (DBA) is a congenital macrocytic anaemia. To investigate whether DBA is due to hyporesponsiveness to or hypoproduction of Steel factor (SF), we compared the in vitro responsiveness of the BFU-E contained in the Ficoll-Hypaque non-adherent cell fraction of six DBA marrows with that of four normal marrows and one transient erythroblastopenia of childhood (TEC) marrow. In addition, we studied the effect of soluble SF on long-term marrow cultures (LTMC) and analysed the stromal cells from these cultures for SF mRNA transcripts. All the patients showed an erythropoietin dose-related increase of small BFU-E. The number and size of BFU-E was increased with the addition to the epo of IL-3 or SF; IL-3+SF was not synergistic. The addition of soluble SF to LTMC of DBA patients was associated with a small but consistent increase in non-adherent cell production and an increase in the number of progenitors. Messenger RNA from immortalized stromal cell lines of three patients and from primary bone marrow stromal cells of one patient showed the presence of expected SF transcripts by PCR analysis. These results demonstrate that this group of DBA patients responds to SF and produces SF mRNA normally, indicating that SF itself is not involved in DBA pathophysiology. The effects observed suggest that, despite the lack of evidence for a causative role, SF may prove to be effective treatment for such patients.

Fanconi's anemia is an autosomal recessive disorder with a high incidence (greater than 90%) of aplastic anemia and a premalignant component with a greater than 10% risk of leukemia or solid tumors. The diagnosis of Fanconi's anemia depends on increased chromosomal breakage in lymphocytes following treatment with a DNA cross-linking agent; patients have been identified who are clinically well and whose physical appearance is normal. Although bone marrow or cord blood transplants can be curative, treatment for the aplastic anemia usually depends on androgens. Close to 20 patients with Fanconi's anemia have delivered normal babies, and the mothers' hematologic status was not significantly adversely affected by the pregnancy. A few patients have clonal cytogenetic abnormalities in their bone marrow that do not necessarily indicate leukemic transformation, but further follow-up is important. Studies of in vitro erythropoiesis indicate a correlation between the clinical hematologic status and the presence of erythroid progenitors in the blood or bone marrow. Certain hematopoietic growth factors do increase growth in vitro, suggesting that new types of therapy may become available. Not every patient has a poor prognosis. There are now many adults with Fanconi's anemia, some with families of their own.

Pure red cell aplasia (PRCA) is a rare disorder of erythrocyte production which is believed to have an autoimmune basis in most cases. Diamond-Blackfan anemia (DBA) is one type of congenital PRCA. Since PRCA has been reported to respond to intravenous gamma-globulin (IVGG) therapy, we administered IVGG to a 2 year old girl with DBA resistant to corticosteroids and observed slight therapeutic effect.

Mitochondrial DNA (mtDNA) deletion is associated with a variety of clinical entities. In addition to progressive external ophthalmoplegia and Kearns-Sayre syndrome, mtDNA deletions have been demonstrated in Pearson's syndrome. We report an mtDNA deletion in an infant with a variant of Pearson's syndrome. Not only does she have congenital anemia, severe tubulopathy, and exocrine pancreas insufficiency, but she also has diabetes and cerebral atrophy. However, there are no signs of gut or liver involvement. Bone marrow improved while new tissues were involved, thus showing variability in progression of the disease. Decreased respiratory chain enzyme activities were demonstrated in muscle, and an mtDNA deletion was demonstrated in muscle, kidney, leukocytes, and fibroblasts.

Crosslink repair of mitomycin C-induced interstrand crosslinks was studied in exponentially growing and confluent normal human, transformed W138CT-1, Fanconi's anemia (FA) and xeroderma pigmentosum (XP) group-A fibroblasts by the assay methods of alkaline sucrose centrifugation, hydroxyapatite column chromatography and S1-nuclease digestion. These three methods demonstrated unequivocally that crosslinking occurred at a rate of 0.13 crosslinks/10(8) Da per microgram per ml mitomycin C (less than or equal to 10 micrograms/ml) and the first half-excision of crosslinks followed the rapid first-order kinetics of 2-3 h half-life in exponentially-growing normal, WI38CT-1 and XP group-A cells. However, the first half-excision was completely defective in three out of the four FA strains tested and severely retarded in an FA strain. These results strongly support our previous observations in different strains of normal human, FA and XP group-A cells. An important new addition is that confluent, otherwise proficient, normal and XP cells almost completely lost the ability of the first, rapid half-excision of mitomycin C crosslinks in their DNA. This probably suggests that the enzyme or regulatory factor responsible for the half-excision, which differs from that for nucleotide excision repair, present constitutively in confluent cells, may be induced or activated only in the cycling cells. However, its relation to a defective FA factor is not clear at present.

Chromosome and sex chromatin studies have contributed significantly to the evaluation of human bone marrow transplants. In addition to their use in documenting bone marrow engraftment following transplantation, they have been used to (1) evaluate whether recurrent leukemia occurs in donor or recipient cells and whether recovery in aplastic anemia results from growth of host or donor cells, (2) demonstrate the bone marrow origin of pulmonary macrophages and hepatic Kupffer cells, (3) show that bone marrow fibroblasts have an origin different from that of the hematopoietic bone marrow cells, (4) evaluate twin zygosity in preparation for transplantation, and (5) show that the defect in Fanconi's anemia is intracellular.

The effects of natural and synthetic androgens on erythroid colony formation in children's bone marrow cultures were studied using a methylcellulose microculture assay. In an attempt to predict the clinical response to androgens in two children with Fanconi anaemia (FA) and two children with Diamond-Blackfan syndrome (DB), we tested the hormonal stimulation of testosterone, nortestosterone and etiocholanolone on CFU-E, BFU-E and uroporphyrinogen I synthase activity (UROS). We observed that colony formation and UROS activity were reduced when compared to values obtained with normal children's bone marrow cultures. The addition of steroids to the cultures significantly enhanced the numbers of CFU-E and BFU-E derived colonies and their UROS activity in marrow from patients with FA and one patient with DB. The strong depletion of marrow progenitor cells in the unresponsive marrow from child 4 with DB could explain the absence of hormonal response. Whereas the responsiveness to steroids varied according to the individual, the in vitro testing of erythroid differentiation in the presence of androgens theoretically may lead to an effective prediction of response to therapy in children with hypoplastic anaemia.

Chromosome modifications induced by BrdU allow a study of the cell division cycle during, at least, three consecutive generations. Among normal lymphocytes, the cell cycle varies in duration, but for the majority of cells, is a little shorter than twenty hours. In trisomy 21 individuals, the mean cell cycle is shorter, around 16 hours. In Fanconi's anemia, the cell cycle is slowed down and its duration is approximatively twice that of normal individuals. Endoreduplicating cells seem to have the cycle of longest duration.

Long-term lymphoid cell lines (LCL) derived from normal individuals, patients with ataxia telangiectasia (A-T), xeroderma pigmentosum (XP), and Fanconi anemia (FA) were exposed to various concentrations of 11 chemical clastogens. The agents were chosen to represent a variety of suggested modes of action. In contrast to all other genotypes, the FA lines demonstrated significant rates of spontaneous chromosomal breakage and showed hypersensitivity to all of the clastogens employed. Variability among lines within a genotype suggested individual responses to specific agents. Computation of "corrected values" to address the problem of baseline disparity removed some of the significant differences between the FA and other lines. Nonetheless, following correction, the FA genotype was still delineated by clastogens which are not DNA cross-linkers. The A-T lines were specifically identified by the induction of chromosome damage by bleomycin and neocarzinostatin.

Hematopoietic stem cell cultures have greatly advanced our understanding of the physiology of blood production at its early stages and have facilitated the study of various factors that regulate cell differentiation and proliferation. With this understanding, the author discusses hematopoiesis, hematopoietic stem cells and assays for their study, and hematopoietic failure. This is followed by a discussion of different forms of bone marrow failure and guidelines for a rational basis for their therapy.

Survival curves were determined for several human diploid fibroblast strains treated with mitomycin-C (MMC) with or without concomitant incubation with superoxide dismutase (SOD). These included cells from patients with Fanconi's anemia (FA), dyskeratosis congenita (DC) a disorder related to FA, Gardner's syndrome (GS) and xeroderma pigmentosum grop C (XPC). Incubation with SOD had no effect on MMC-induced cytotoxicity in the two normal cell strains, XPC or GS. Although GM2053 (FA) and two DC strains showed intermediate sensitivity to killing by MMC similar to XPC and GS, the survival of these cell strains was increased approximately 2-4 times after 0.5 microgram/ml of MMC by concomitant treatment with SOD. Survival of the most MMC sensitive cell strain GM1309 (FA) was sharply increased by adding SOD; survival was enhanced about 15-fold after treatment with 0.1 microgram/ml of MMC. SOD induced no enhancement of survival in MMC treated normal cells at survival levels down to nearly 10(-3). We propose that the hypersensitivity to MMC cytoxicity in FA and DC cells may involve among other factors a deficiency in the inactivation of certain free radical intermediates.

Ferrokinetic studies were carried out in 4 patients with Fanconi's anaemia (FA). Experimental data were analysed by means of a mathematical model of iron kinetics in order to obtain a quantitative assessment of effective and ineffective erythropoietic activity, mean red cell lifespan, and non-erythroid iron turnover. The major pathogenetic mechanism of the anaemia appeared to be relative marrow failure, i.e. a reduction in the proliferative capacity of the erythroid marrow. The role of ineffective erythropoiesis was of minor importance. On the basis of the results obtained both the pathogenesis and the natural course of the disease are discussed.

Three children with Fanconi's anemia belonging to a family where 6 children had the disease were investigated. One child had growth hormone deficiency, a second child showed subnormal response of testosterone to gonadotropin stimulation and the third child had a missing insulin release following arginine. This report shows that growth hormone deficiency is not necessarily liniked with Fanconi's anemia when it occurs in a family. Multiple endocrine insufficiencies do no appear to be part of the disease.

Serial haematological investigations were carried out in 5 patients with Fanconi's anaemia over periods of 6 months--11 years. All the patients were pancytopenic with a depression of the granulocytic and megakaryocytic elements of the bone marrow throughout the greater part of their illnesses. Erythropoietic acitvity was variable. The initial bone marrow examination revealed depressed erythroid function in 3 patients. The erythroid hypoplasia persisted in 2 of them, while in the third, erythroid activity increased with time, possibly as the result of therapy with oxymetholone. Erythroid hyperplasia was present in the remaining 2 patients, both at presentation and throughout the course of the illness. This could not be attributed to treatment in either patient. Six erythrokinetic studies were carried out in the 5 patients at variable intervals after the diagnosis had been made. In 2 studies erythroid activity was unequivocally depressed, while in a further 3 a significant, though probably suboptimal, erythroid marrow response was present. In the final study erythropoiesis was increased but was markedly ineffective in terms of the delivery of viable red cells into the circulation. In vivo counting suggested that some degree of ineffective erythropoiesis was also present in the other patients and studies with 51Cr indicated a shortened red cell survival in all subjects studied. In 2 of them significant splenic sequestration was present. Leukaemic transformation occurred in 2 patients. In 1 of them its development was heralded by the appearance of micromegakaryocytes in the bone marrow.

In order to develop the usefulness of Fanconi's anemia (FA) lymphoblast lines for biochemical and genetic studies, we have determined their sensitivity to a variety of DNA-damaging chemicals. We have adapted a growth inhibiton protocol in which the sensitivity of a cell line is characterized by the drug concentration yielding a 50% inhibiton of growth (EC50). The DNA-cross-linking agents, mitomycin C, nitrogen mustard, melphalan, 1,3-butadiene diepoxide, cis-diaminedichloroplatinum(II), and cyclophosphamide, were all more toxic to four FA cell lines than to five normal lines. Three lines, HSC 72 (FA), 99 (FA) and 230 (FA), had EC50s that were 10 to 20 times lower than that of controls while the fourth line, HSC 62 (FA), had an intermediate EC50. Three nitrosourea compounds were also more toxic to FA cells than to controls. However, 2 normal cell lines (HSC 92 and 93) had nitrosourea EC50s 4 to 7 times lower than the other nine controls and overlapped the sensitivity of the intermediate [HSC 62 (FA)] cell line. The same 2 normal cell lines were also more sensitive than 12 other controls, including FA heterozygotes, xeroderma pigmentosum, and ataxia telangiectasis, to the monofunctional alkylating agents, ethyl methane sulfonate, methyl methane sulfonate, and N-methyl-N'-nitro-N-nitrosoguanidine. Heterogeneity was also found with FA lines. Two FA cell lines (HSC 72 and 230) had EC50s lower than all control lines while one FA line (HSC 99) had an EC50 similar to that of the resistant normal lines. FA and normal cells had nearly the same sensitivity to 4-nitroquinoline-1-oxide and bleomycin. These results demonstrate that FA lymphoblast lines are more sensitive than normal cell lines to all DNA-cross-linking agents examined. These cell lines should therefore be useful for the analysis of DNA cross-link repair and the biochemical defect in FA. We have also found an unexpected sensitivity of some FA and normal lines to monofunctional alkylating agents.

The lifespan of fibroblasts from genetic syndromes with reduced DNA repair or chromosome stability has been measured. Cells from Bloom's syndrome, Cockayne's syndrome, Fanconi's anaemia and 2 out of 3 cases of ataxia telangiectasia had a significantly reduced growth potential in comparison to controls. In each case the longevity of several parallel populations was measured and the greatest variability in lifespan was observed with Cockayne's syndrome cells. The fibroblasts from 1 ataxia telangiectasia patient and a Friedreich's ataxia patient grew to the passage levels seen in control cultures. The results suggest that repair processes are necessary for cells to achieve their maximum in vitro lifespan, and support the error theory rather than the programme theory of ageing.