Fanconi anemia (FA) is a rare genetic disease discovered 80 years ago by Guido Fanconi, an eminent Swiss pediatrician. It is characterized by short stature, skeletal anomalies, increased incidence of solid tumors and leukemias, bone marrow failure and cellular sensitivity to DNA damaging agents. Following a historical account, exemplary case reports and the current status of FA genes and their mutations, this volume discusses neoplasia in FA as well as current approaches to pre- and postnatal diagnosis. Further topics include revertant mosaicism as a kind of 'natural gene therapy' and hematopoietic stem cell transplantation as the only curative approach in FA. The final chapters investigate evolutionary aspects of the FA genes with special emphasis on the avian genome and the involvement of FA genes in recombinational types of DNA repair. Physicians and researchers in the fields of pediatrics, hematology, cancer, genetics, DNA repair and aging will benefit from understanding this disease, which illustrates the complex network of genomic maintenance systems that protect us from cancer and premature aging.

Incorporation of bromodeoxyuridine (BrdU) during DNA replication is frequently used for cell cycle analysis. The flow cytometric BrdU/Hoechst quenching technique is conducive to high-resolution assessment of cell cycle kinetics, but requires continuous BrdU treatment, which may have cytostatic or cytotoxic effects. Here, we have examined the impact of BrdU on the proliferation of BT474 and SK-BR-3 breast cancer cell lines and compared the observed effects with cell proliferation of RT4 and J82 bladder carcinoma cells, previously described to be sensitive and insensitive to BrdU, respectively. Both uni- and bi-parametric DNA measurements were performed to identify BrdU-induced alterations in the S-phase fraction and in cell cycle progression. An annexinV/propidium iodide (PI) assay was used to identify potential induction of apoptosis by BrdU. Proliferative activity in BT474, SK-BR-3, and RT4 cultures was reduced in different cell cycle phases due to continuous treatment with 60, 5.0, and 3.5 micro m BrdU. This effect, which was not found in J82 cultures, was dependent on exposure time (96 versus 48 h) and was also dose-dependent for RT4 and SK-BR-3. BrdU application does not induce apoptosis or necrosis as revealed with the annexin V/PI assay. We concluded that continuous BrdU treatment did not affect cell viability, but essentially alters cell cycle progression in three out of four cell lines tested. Cell-type specific validation of the feasibility of the powerful BrdU/Hoechst quenching technique is required and recommended.

Human mid-trimester amniotic fluid cells were cultivated under conditions of decreased oxygen supply. Compared to control cultures the low-oxygen group showed improved growth which was quantitated by three independent assays (1) direct cell counts, (2) bromodeoxyuridine (BrdU)-Hoechst flow-cytometry, and (3) cloning efficiency. The growth promoting effects of lowered oxygen hold for all major morphologic categories of amniotic fluid cells.

Cellular gene expression changes during ontogenetic development of cell physiological activation-inhibtion and differentiation. Classical molecular assays like Southern, Northern, or Western blotting display only a few genes at once. The analysis of complex alterations of gene expression patterns, therefore, requires large quantities of biological materials, has significant experimental inter- and intravariability, and is quite time-consuming. Some of these problems became negligible with the advent of microarray techniques (,). cDNAs or oligonucleotids are immobilized on glass or membrane surfaces, cDNA or cRNA transcribed from cellular mRNA is hybridized, and signal detection is performed by radioactive or fluorescent techniques. The expression of up to several tens of thousands of genes can be made visible on small arrays, enabling investigators a rapid quantitative and qualitative analysis of pro- or eukaryotic gene expression patterns.

We optimized a novel nonradioactive hybridization technique using a cis-platin coupled digoxigenin derivative for direct labeling of mRNA. This new mRNA reporter molecule was applied to cDNA membrane arrays to simultaneously identify expression of hundreds of genes. The sensitivity of this nonradioactive mRNA hybridization technique was comparable to radioactive cDNA labeling on house-keeping gene expression but even superior on the detection limit of the expression of low abundant genes using mRNA isolated from human diploid fibroblasts. Additional advantages are faster readout and decreased total working times because of luminescence technology and avoidance of radioactivity. Finally, no potential artifactual reverse transcription step is necessary because of direct labeling of mRNA used for hybridization on nucleic acid arrays.

We applied the cDNA differential display technique (DDT) in a DNA-repair deficient cell model to isolate genes involved in dysregulation of cell proliferation and development of cancer. The comparative analysis of mRNA expression patterns of human diploid fibroblasts from Fanconi's amemia (FA) and normal phenotype led to the identification of a novel cDNA CO9. Northern blot analysis reveals that CO9 is significantly upregulated in FA fibroblasts but downregulated or absent in fibroblasts from normal donors. CO9 was also highly expressed in FA B-cells of complementation group A and in Raji cells. However, CO9 is not expressed in FA complementation groups B, C, D and E. The full-length cDNA is 840bp long and contains an open reading frame of 216bp (72 amino acids), which encodes for a 7.6-kDa protein. The lengths of the 5′ and 3′ untranslated region are 165 and 459bp, respectively. The N-terminal and C-terminal nucleotide sequence of CO9 shows homology to a putative human l-3-phosphoserine phosphatase identified recently (HSPSPASE, EMBL Accession No. Y10275) but lacks a 476-bp stretch in the open reading frame. The loss of nucleotides within the open reading frame introduces a new termination codon in the CO9 cDNA along with a novel COOH terminus resulting in a new protein product. Database chromosome mapping localized CO9 to chromosome 7q 11.2. We hypothesize that CO9 represents a novel protein being a partial homologue to the l-3-phosphoserine phosphatase but with a different regulatory cell function.

The differential display technique (DDT) was used to compare Fanconi anaemia (FA) fibroblasts with those of normal controls in a screen for genes involved in DNA repair, recognizing and handling damage or indicating cell cycle abnormalities as a result of genetic changes. The DDT revealed two different deletions of 5 and 11 bp at a single locus in the 3' untranslated region (UTR) of a gene known to encode human alpha-tropomyosin (TPM1) in FA cells. These small deletions were detected by analysis of shifted 900-bp long cDNA fragments on polyacrylamide gels. They were characterized as loss of GTTTT or TGTTTTGTTTT, respectively, in a region with five GTTTT tandem repeats. Since it was postulated that the 3' UTR of the TPM1 gene plays a regulatory role in cell differentiation and tumour suppression, the existence and possible patterns of deletions in a variety of normal donors was investigated. The heterogenous distribution of non-deleted, 5- and 11-bp deleted 3' UTR regions indicate a polymorphism of the TPM1 gene in this tandem repeat motif. Therefore the expression pattern of these mutations among FA and non-FA cells rendered any direct relationship to the putative DNA repair defect in FA unlikely. Of note, however, the fact remains that such deletions reportedly facilitate mRNA degradation and may bear significance in the TPM1 gene action. Finally, of further interest is the finding that even small deletions can be identified by DDT in addition to the identification of the differential expression patterns of genes.

Control of successful genetic complementation of cellular defects in heterogenous cell populations requires biochemical selection markers or cell analytical specifiers. With available gene therapy technologies, only a fraction of a cell population is transfected or transduced. We applied and optimized a novel dual-laser flow cytometric technique to analyze immediately the genetic complementation of cells dysregulated in cell proliferation and DNA repair. A novel bicistronic retrovirus carrying the normal Fanconi anemia gene (group C) and the cell surface marker gene l-NGFR was constructed to analyze the normalization of the G2-phase cell cycle defect in DNA-repair-deficient FA(C) lymphoblastoid cells after transduction. Using a dual-laser multiparameter technology, we 1) analyzed the cell-cycle distribution of viable/dead cells using Hoechst 33342 (with ultraviolet light), 2) identified the genetically complemented cells by FITC antibody labeling of the novel l-NGFR surface marker (488 nm), and 3) recorded mitomycin C-induced cell death in nontransduced and transduced cells by propidium iodide. Artificial l-NGFR expression is high and similar in ontogenetically and phylogenetically different cell populations. With this novel l-NGFR marker technology, the success of gene therapy of cell-cycle dysregulation in even small subpopulations of cells can be recorded within 48 h. In addition to improved cell analysis, l-NGFR surface marker expression can also be used for rapid generation of pure cell populations by column cell separation technologies.

Conventional subtraction library techniques or DNA-transfection studies are standard techniques applied for identification and isolation of genes relevant in monogenetic diseases like Fanconi anemia (FA). The differential display technique (DDT) was developed to compare mRNA expression between a mutant cell line and its syngeneic control and allows comparison of almost all mRNA species within a short time. However, for identification of genes relevant in monogenetic diseases, no syngeneic cell model is available. In this report, we show that the use of nonsyngeneic diploid human fibroblasts does not increase the number of differentially displayed bands due to diversity of untranslated regions. cDNA bands with a length of up to 1000 bp were obtained and applied to DDT. After screening of about 13000 cDNA bands, only 0.5% were found to be differentially expressed between FA and control cells. Finally, three mRNAs were cloned and verified in Northern blot experiments to be differentially expressed in FA fibroblasts. The low number of differentially displayed cDNA bands in DDT indicates the usefulness of this statistical, molecular approach for identification of multiple genes dysregulated in gene regulation cascades potentially relevant for cell cycle disturbances.

Retroviral vectors are effective shuttle systems by introducing therapeutically relevant genes stably into the genome of proliferating cells. The majority of vectors applied for research or clinical applications use neomycin for cell selection and identification. To circumvent the time consuming and potentially toxic G418 selection process in transduction studies we constructed a novel marker vector using l-NGFR as a cell surface marker to identify DNA repair defective Fanconi anemia cells complemented with the FAC gene. The new vector constructed is based on a MoMLV backbone, a signal peptide-deleted l-NGFR receptor gene under control of a LTR promoter and the therapeutically relevant FAC gene placed downstream of a SV40 promoter. Supernatants containing high titers of amphotropic viruses from FACS cloned cell cultures were obtained and tested for primary transduction rates, rapid detection of transduced cells within 48 h and correction of mitomycin C-induced cell cycle G2 phase accumulation in a single assay using multiparameter, dual laser flow cytometry. Primary transduction efficiency detected via 〈l-NGFR〉 antibody was between 5% and 30% with Fanconi cell lines, 5% with CD34+ cells and 15% with PBLs. MMC-induced G2 phase cell cycle disturbances were fully complemented in Fanconi anemia B cell lines of complementation group C but not in B cell lines of another FA complementation group (D). In addition to the normalization of the G2 phase arrest, induction of cell death in the FAC cell line was also decreased three- to 10-fold at different MMC concentrations.

The effects of the tear gas 2-chlorobenzylidene malonitrile (CS) on mammalian cell proliferation were studied in detail using bromodeoxyuridine/Hoechst flow cytometry. In synchronized (G0/G1-phase) Chinese hamster embryo (CHE) cells, exposure to CS (60 microM) caused a permanent arrest in the G0/G1 phase in 50% of the cells and a delayed G0/G1 phase exit. In asynchronously growing CHE cells, the CS-induced cell kinetic perturbations varied with the cell cycle stage during treatment. While G1-phase cells showed a delayed progression through S and G2/M phases, S-phase cells were mainly inhibited in the G2/M compartment of the first cell cycle. In contrast, CS-treated, asynchronous, amniotic fluid-derived, fibroblast-like (AFFL) cells exhibited a prolonged transit through the G2/M phase of the first cell cycle regardless of the cell cycle stage during treatment. This indicates that the induced cytotoxicity of CS is a function of both the cell cycle phase and the particular type of cells.

The effect of the tumor promoter okadaic acid on cell cycle progression and on vimentin expression in MPC‐11 mouse plasmacytoma cells was compared with that of the tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). Cell cycle progression of asynchronously grown MPC‐11 cells was inhibited by both agents, but, in contrast to the G1 phase arrest caused by TPA, okadaic acid gave rise to G2/M phase and S phase arrest. This effect of okadaic acid was delayed significantly compared to the TPA‐caused arrest. Furthermore, okadaic acid was able to induce vimentin expression to an extent comparable to the TPA response. However, vimentin expression was markedly delayed in okadaic acid‐treated relative to TPA‐treated cells. Another protein phosphatase inhibitor, calyculin A, also induced cell cycle changes and vimentin expression at concentrations at or above 1 × 10−9M. Based on these observations, we suggest an involvement of protein phosphatase 1 (possibly also phosphatase 2A and/or other phosphatases) in both the G2/M cell cycle block and the induction of vimentin expression in MPC‐11 cells by okadaic acid. © 1995 Wiley‐Liss, Inc.

High resolution, multiparameter analysis using the flow cytometric BrdU/Hoechst quenching technique has been applied to study cell cycle kinetics and vimentin expression in individual cells of asynchronously grown MPC‐11 mouse plasmacytoma cell cultures treated with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) to induce in vitro differentiation. BrdU treatment up to 16 h in the absence or presence of TPA did not affect either cell cycle progression or the kinetics or quantity of vimentin expression. TPA‐treated cells became arrested in G1 phase of the second cell cycle; however, this G1 phase arrest was transient only. In addition, G1 phase cells located prior to a putative transition point at the beginning of TPA treatment were completely blocked in cell cycle progression. There is also evidence that cells located in G1 or G2/M phase at the beginning of TPA treatment finally expressed low levels of vimentin. On the contrary, cells located in S phase at TPA exposure showed high vimentin levels after treatment. The results presented here show that, with the flow cytometric BrdU/Hoechst quenching technique, one can correlate time‐dependent protein expression at the single cell level in asynchronously grown cultures not only with the actual cell cycle state, but also with the history of cell replication. © 1994 Wiley‐Liss, Inc.

A monoclonal antibody (Ki-S1) has been raised that reacts with the nuclei of proliferating cells. The antigen recognized is resistant to formalin fixation and can be detected in frozen tissues as well as in routinely processed specimens. In immunohistochemistry, nuclear staining can be seen in those tissues and cellular compartments known to be actively proliferating. Peripheral blood lymphocytes are negative but show a strong increase in antigen expression after mitogen stimulation. Flow cytometric determination of DNA content and antigen expression revealed negativity of G0 cells and positivity of G1 to G2/M cells. A cytoplasmic co-reactivity, not associated with proliferation, was confined to Langerhans islands of the pancreas. The nuclear localized antigen has a molecular mass of 160 kd and therefore seems to be different from all other known immunohistochemical markers of proliferating cells. We conclude that the monoclonal antibody Ki-S1 might provide a useful tool for studying cell proliferation in situ under normal and pathological circumstances.

Continuous labelling of cells with deoxybromouridine (BrdUrd) followed by staining with a bis-benzimidazole (Hoechst 33258) and a phenanthridinium (propidium iodide or ethidium bromide) allows the cells to be separated by flow cytometry according to the extent of their DNA replication. This BrdUrd-Hoechst/PI method has been used mainly to observe perturbations of the cell cycle in synchronously growing cells. In this paper we demonstrate that, when the method is applied to asynchronously dividing cells, more extensive information can be derived about the effects of cytotoxic and other treatments on the kinetics of the cell cycle. The interpretation of the data is explained, the effects of different types of cytotoxic agent are described, and the method is compared briefly to other methods for following cell cycle kinetics.

We correlated cell cycle progression and vimentin expression at the single cell level by multiparameter flow cytometry in populations of MPC-11 cells enriched in different cell cycle phases by centrifugal elutriation and subsequently treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Synchronized, untreated cultures showed a uniform, synchronous progression through the cell cycle during further cultivation. A 6-h TPA treatment of G1-phase-enriched cultures induced both a partial G1-phase arrest in the same cycle and a moderate fraction of cells to become vimentin positive. However, nearly all cells of the cultures enriched in S- or in G2/M-phase cells could be arrested by TPA treatment at the earliest in the G1 phase of the second cell cycle and displayed higher fractions of positive cells as well as higher average levels of vimentin. After 20 h of treatment, the G1-phase arrest was almost complete. In terms of fractions of vimentin-positive cells as well as of average cellular vimentin content, the differences between the cultures resembled, albeit on a higher level, those between the respective cultures treated with TPA for 6 h. These observations might explain the striking bimodal distribution of individual cellular vimentin content detectable in G1-phase fractions of asynchronous, TPA-treated cultures. The pattern of vimentin mRNA accumulation in synchronized cultures after short-term TPA treatment strongly suggests that the cell cycle-dependent pattern of vimentin expression is caused, at least in part, by different levels of vimentin mRNA accumulated in the cells. Since proteinaceous mediator(s) are obviously involved in TPA-induced vimentin expression in MPC-11 cells, cell cycle-dependent vimentin expression in these cells may be dependent on cell cycle-dependent regulation of the activity and/or concentration of such mediator(s).