Mismatch repair gene defects in sporadic colorectal cancers with microsatellite instability.

Microsatellite instability has been observed in both sporadic and hereditary forms of colorectal cancer. In the hereditary form, this instability is generally due to germline mutations in mismatch repair (MMR) genes. However, only one in ten patients with sporadic tumours exhibiting microsatellite instability had a detectable germline mutation. Moreover, only three of seven sporadic tumour cell lines with microsatellite instability had mutations in a MMR gene, and these mutations could occur somatically. These results demonstrate that tumours can acquire somatic mutations that presumably do not directly affect cell growth but result only in genetic instability. They also suggest that many sporadic tumours with microsatellite instability have alterations in genes other than the four now known to participate in MMR.

Founding mutations and Alu-mediated recombination in hereditary colon cancer.

By screening members of Finnish families displaying hereditary nonpolyposis colorectal cancer (HNPCC) for predisposing germline mutations in MSH2 and MLH1, we show that two mutations in MLH1 together account for 63% (19/30) of kindreds meeting international diagnostic criteria. Mutation 1, originally detected as a 165-base pair deletion in MLH1 cDNA comprising exon 16, was shown to consist of a 3.5-kilobase genomic deletion most likely resulting from Alu-mediated recombination. Mutation 2 destroys the splice acceptor site of exon 6. A simple diagnostic test based on polymerase chain reaction was designed for both mutations. Our results show that these two ancestral founding mutations account for a majority of Finnish HNPCC kindreds and represent the first report of Alu-mediated recombination causing a prevalent, dominantly inherited predisposition to cancer.

Analysis of mismatch repair genes in hereditary non-polyposis colorectal cancer patients.

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant disorder characterized by the early onset of colorectal cancer and linked to germline defects in at least four mismatch repair genes. Although much has been learned about the molecular pathogenesis of this disease, questions related to effective presymptomatic diagnosis are largely unanswered because of its genetic complexity. In this study, we evaluated tumors from 74 HNPCC kindreds for genomic instability characteristic of a mismatch repair deficiency and found such instability in 92% of the kindreds. The entire coding regions of the five known human mismatch repair genes were evaluated in 48 kindreds with instability, and mutations were identified in 70%. This study demonstrates that a combination of techniques can be used to genetically diagnose tumor susceptibility in the majority of HNPCC kindreds and lays the foundation for genetic testing of this relatively common disease.

Regulation of proliferation and cytokine expression of bone marrow fibroblasts: role of c-myb.

The c-myb protooncogene plays a major role in regulating the process of in vitro and in vivo hematopoiesis via its activity as transcriptional regulator in hematopoietic progenitor cells. Since the bone marrow microenvironment appears to regulate in vivo hematopoiesis by maintaining the growth of multipotent progenitors via secretion of specific cytokines, we asked whether c-myb is also required for the proliferation of and/or cytokine production by stromal cells that generate fibroblast-like colonies (fibroblast colony-forming units [CFU-F]). Using the reverse transcriptase polymerase chain reaction technique, we detected low levels of c-myb mRNA transcripts in human normal bone marrow fibroblasts. Treatment of these cells with c-myb antisense oligodeoxynucleotides caused downregulation of c-myb expression, decreased in the number of marrow CFU-F colonies (approximately 54% inhibition) and in the cell number within residual colonies (approximately 80%), and downregulation of granulocyte/macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF) mRNA expression. Transfection of T98G glioblastoma cells, in which expression of c-myb, GM-CSF, and SCF mRNAs is undetectable or barely detectable, with a plasmid containing a full-length c-myb cDNA under the control of the SV40 promoter induced the expression of biologically active SCF and GM-CSF in these cells. Regulation of GM-CSF expression by c-myb was due in part to transactivation of the GM-CSF promoter. These results indicate that, in addition to regulating hematopoietic cell proliferation, c-myb is also required for proliferation of and cytokines synthesis by bone marrow fibroblasts.

Skin lipids: their biochemical uniqueness.

Two key words characterize the uniqueness of skin lipids: complexity and perversity. Each suggests a function. Complexity manifests itself in the large number and variety of both saturated and unsaturated fatty chains synthesized by human skin. Functionally, this allows each individual to have a distinct odor or chemical fingerprint. Perversity manifests itself when one compares the lipids synthesized by skin with those synthesized by internal tissues. For example, skin makes odd instead of only even chains, branched instead of only straight chains, free instead of only esterified acids, places double bonds in unusual positions in the fatty chains, extends chains to extreme lengths, and accumulates intermediates in the synthesis of a biologically valuable compound such as cholesterol. Functionally, these products may pose metabolic problems to potential pathogens and thus contribute to the survival of only compatible microorganisms.

Selective inhibition of leukemia cell proliferation by BCR-ABL antisense oligodeoxynucleotides.

To determine the role of the BCR-ABL gene in the proliferation of blast cells of patients with chronic myelogenous leukemia, leukemia blast cells were exposed to synthetic 18-mer oligodeoxynucleotides complementary to two identified BCR-ABL junctions. Leukemia colony formation was suppressed, whereas granulocyte-macrophage colony formation from normal marrow progenitors was unaffected. When equal proportions of normal marrow progenitors and blast cells were mixed, exposed to the oligodeoxynucleotides, and assayed for residual colony formation, the majority of residual cells were normal. These findings demonstrate the requirement for a functional BCR-ABL gene in maintaining the leukemic phenotype and the feasibility of gene-targeted selective killing of neoplastic cells.

Mutations of GTBP in genetically unstable cells.

The molecular defects responsible for tumor cell hypermutability in humans have not yet been fully identified. Here the gene encoding a G/T mismatch-binding protein (GTBP) was localized to within 1 megabase of the related hMSH2 gene on chromosome 2 and was found to be inactivated in three hypermutable cell lines. Unlike cells defective in other mismatch repair genes, which display widespread alterations in mononucleotide, dinucleotide, and other simple repeated sequences, the GTBP-deficient cells showed alterations primarily in mononucleotide tracts. These results suggest that GTBP is important for maintaining the integrity of the human genome and document molecular defects accounting for variation in mutator phenotype.

Mutation of a mutL homolog in hereditary colon cancer.

Some cases of hereditary nonpolyposis colorectal cancer (HNPCC) are due to alterations in a mutS-related mismatch repair gene. A search of a large database of expressed sequence tags derived from random complementary DNA clones revealed three additional human mismatch repair genes, all related to the bacterial mutL gene. One of these genes (hMLH1) resides on chromosome 3p21, within 1 centimorgan of markers previously linked to cancer susceptibility in HNPCC kindreds. Mutations of hMLH1 that would disrupt the gene product were identified in such kindreds, demonstrating that this gene is responsible for the disease. These results suggest that defects in any of several mismatch repair genes can cause HNPCC.

Positive autoregulation of c-myb expression via Myb binding sites in the 5' flanking region of the human c-myb gene.

The nuclear proto-oncogene c-myb is preferentially expressed in lymphohematopoietic cells, in which it plays an important role in the processes of differentiation and proliferation. The mechanism(s) that regulates c-myb expression is not fully understood, although in mouse cells a regulatory mechanism involves a transcriptional block in the first intron. To analyze the contribution of the 5' flanking sequences in regulating the expression of the human c-myb gene, we isolated a genomic clone containing extensive 5' flanking sequences, the first exon, and a large portion of the first intron. Sequence analysis of a subcloned 1.3-kb BamHI insert corresponding to 687 nucleotides of the 5' flanking sequence, the entire first exon, and 300 nucleotides of the first intron revealed the presence of closely spaced putative Myb binding sites within a segment extending from nucleotides -616 to -575 upstream from the cap site. A 165-bp segment containing these putative Myb binding sites was linked to a human thymidine kinase (TK) cDNA driven by a low-activity proliferating cell nuclear antigen promoter and cotransfected into TK- ts13 cells with a plasmid in which a full-length human c-myb cDNA is driven by the early simian virus 40 promoter; Myb inducibility of TK mRNA expression was observed both in transient expression assays and in stable transformants. The highest level of inducibility was detected when the 165-bp fragment was placed 138 bp upstream of the proliferating cell nuclear antigen promoter-TK cDNA reporter unit or 3' of the TK cDNA. Mutation of the putative Myb binding sites greatly reduced c-myb transactivation of TK mRNA expression and specifically reduced the binding of in vitro-translated Myb protein at those sites. Finally, c-myb transactivated TK mRNA expression driven by a segment of the authentic c-myb 5' flanking region containing the Myb binding sites. These data suggest that human c-myb maintains high levels of Myb protein in cells that require this gene product for proliferation and/or differentiation by an autoregulatory mechanism involving Myb binding sites in the 5' flanking region.

A naturally occurring hPMS2 mutation can confer a dominant negative mutator phenotype.

Defects in mismatch repair (MMR) genes result in a mutator phenotype by inducing microsatellite instability (MI), a characteristic of hereditary nonpolyposis colorectal cancers (HNPCC) and a subset of sporadic colon tumors. Present models describing the mechanism by which germ line mutations in MMR genes predispose kindreds to HNPCC suggest a "two-hit" inactivation of both alleles of a particular MMR gene. Here we present experimental evidence that a nonsense mutation at codon 134 of the hPMS2 gene is sufficient to reduce MMR and induce MI in cells containing a wild-type hPMS2 allele. These results have significant implications for understanding the relationship between mutagenesis and carcinogenesis and the ability to generate mammalian cells with mutator phenotypes.

Correlation between E2F-1 requirement in the S phase and E2F-1 transactivation of cell cycle-related genes in human cells.

The mammalian nuclear protein E2F-1 has recently been cloned based on its ability to bind the retinoblastoma protein. To determine whether E2F-1 plays a role in the control of the cell proliferation, we introduced an inducible construct expressing an E2F-1 antisense RNA into the human glioblastoma T98G cell line and assessed DNA synthesis during the cell cycle. Expression of the antisense transcripts during the G1-S transition resulted in a marked delay in the completion of DNA synthesis. Band-shift analysis of bacterially produced E2F-1 showed that this protein bound to the promoters of human DNA polymerase-alpha, cyclin D1, and c-myb but not to the cdc2 gene promoter. E2F-1 also transactivated the bound promoters in transient transfection assays. These results suggest a major role for E2F-1 in the control of cell cycle progression via transcriptional regulation of proliferation-associated genes.

Retroviral transfer of genes into erythroid progenitors derived from human peripheral blood.

Human erythroblasts are a logical target for studies of expression of transferred globin genes because high-level expression is a prerequisite for gene therapy of hemoglobinopathies. Early erythroid progenitors (erythroid burst-forming units, BFU-E) are readily available from human peripheral blood and can be cultured to produce erythroblasts. However, conditions for efficient transfer into these normal progenitors have not been previously described. Here we demonstrate efficient transfer of the neomycin resistance gene into human peripheral blood BFU-E using the retrovirus vector, N2. We show that liquid culture of mononuclear cells from peripheral blood for 18-24 h prior to retroviral infection leads to increased transfer efficiency of N2 as determined by G418 resistance, and we are able to detect viral DNA by polymerase chain reaction (PCR) analysis. In addition, a second retrovirus, beta(gamma)-SVX, prepared with a human beta-globin gene containing a gamma-globin second exon to facilitate transcript detection and the 3'-enhancer sequence, was also used to determine whether similar results could be obtained when more than one gene is transferred. Using the beta(gamma)-SVX virus, increased transfer efficiency into BFU-E was similarly found after liquid culture for up to 4 days. Expression of the transferred globin gene was also detected by PCR analysis of cDNA made from erythroblast RNA. The human peripheral blood BFU-E system described should allow determination of sequences required for high-level expression of transferred globin and other erythroid genes.

c-myb and growth control.

The available evidence indicates that c-myb plays an important role in the proliferation of hematopoietic cells and in those nonhematopoietic cell types in which c-myb is expressed. A critical aspect in the regulation of c-myb expression rests in the positive autoregulatory mechanism, which is dependent on the interaction of myb protein with the 5' flanking region of the human c-myb gene. The positive autoregulation of c-myb, in conjunction with tissue-specific mechanisms that most likely involve efficient transcription beyond the site of "transcriptional pause" in the c-myb first intron, might allow the generation of c-myb transcripts at levels sufficiently high for optimal biological activity (e.g., at the G1/S transition of the cell cycle). Other transactivating factors, such as the Jun family members, also appear to be involved in regulating c-myb expression. Such factors might act to increase basal levels of c-myb expression to allow activation of the autoregulatory mechanism, or might cooperate with myb in transcriptional regulation of c-myb expression. The function of c-myb is ultimately dependent on the genes that are regulated by the myb product. Preliminary evidence suggests that DNA polymerase-alpha and cdc2, two genes that are critical for DNA synthesis, contain myb binding sites in their promoter region that appear to be required for myb transactivation of their expression. The paradox of the generality of the mechanisms by which c-myb affects cell proliferation and the apparent tissue-specific expression of this gene might be resolved by the growing evidence that the tissue distribution of c-myb is more general than previously appreciated, and that many cell types with no detectable c-myb expression contain a functional equivalent of this gene. For example, B-myb a gene that is homologous to c-myb in the DNA binding and transactivating domains and appears to be ubiquitously expressed, is also required for cell proliferation and, like c-myb, appears to regulate the expression of cdc2, a gene required for cell cycle progression. Together, these findings indicate a general role of members of the myb family in regulation of cell proliferation.

Role of insulin receptor substrate-2 in interleukin-9-dependent proliferation.

Interleukin-9 (IL-9) stimulation results in JAK, STAT and IRS1/2 phosphorylation. The role of IRS adaptor proteins in IL-9 signaling is not clear. We show that IL-9 induces IRS2 phosphorylation and association with phosphatidylinositol-3 kinase (PI 3-K) p85 subunit in TS1 cells and BaF/9R cells, which proliferate upon IL-9 stimulation. We observed a PI 3-K-dependent phosphorylation of protein kinase B (PKB) in TS1 cells, but not in BaF/9R, nor in other IL-9-dependent cell lines. Finally, 32D cells that were transfected with the IL-9 receptor but lack IRS expression survived in the presence of IL-9. Ectopic IRS1 expression allowed for IL-9-induced proliferation, in the absence of significant PKB phosphorylation.

A simple, efficient method for the separate isolation of RNA and DNA from the same cells.

A method for the isolation of total cytoplasmic RNA and high molecular weight DNA from the same cells is described. Cells are gently lysed with NP40 in the presence of vanadyl ribonucleoside complex and the nuclei pelleted by centrifugation. RNA is purified by phenol/CHCl3 extraction of the lysate supernatant followed by ethanol precipitation. Protein is removed from high molecular weight DNA by salt-precipitation after nuclei are digested with proteinase K in the presence of sodium dodecyl sulfate. High yields of clean, intact RNA and DNA are obtained. A major advantage of the method is that it can be scaled down to quantitatively extract RNA and DNA from as little as 1000 cells.

Meibomian gland dysfunction. I. Keratin protein expression in normal human and rabbit meibomian glands.

The expression of keratin proteins from meibomian glands and their correlation with skin epidermal keratins were determined. Keratin proteins were localized in both human and rabbit meibomian glands by indirect immunofluorescence using mouse monoclonal antibodies AE1, AE2 and AE3, which are known to react with human epidermal keratins as well as with keratins from other sources. Keratin proteins from rabbit meibomian glands were further isolated and characterized by SDS-PAGE and immunoblot using mouse monoclonal antibodies AE1 and AE3. Meibomian glands from human and rabbit showed similar immunofluorescent staining with each monoclonal antibody. AE1 antibody, which stains human basal epithelial cells of skin, stains all duct epithelial cells in the human but only the superficial duct epithelial cells in the rabbit meibomian gland. AE2 antibody, which stains human suprabasal epithelial cells of skin and is a marker for fully keratinized epithelia, stains the suprabasal epithelial cells of the central duct and ductules in both the human and rabbit meibomian gland. AE3 antibody, which stains all human epithelial cells of skin, stains all epithelial cells of the duct and ductules, as well as the basal epithelial cells of the acinus in both the human and rabbit meibomian gland. Keratins isolated from whole rabbit meibomian glands contained a 65-67 kD and 58 kD AE3-positive, and a 56.5 kD and 50 kD AE1-positive keratin protein. Expression of 65-67 kD/56.5 kD keratin proteins, and the immunofluorescent staining of the duct epithelium by the AE2 antibody, indicate that the meibomian gland duct epithelium is committed to the process of keratinization.

Meibomian gland studies: histologic and ultrastructural investigations.

Heightened interest in meibomian gland dysfunction has prompted us to evaluate the normal morphological and ultrastructural characteristics of the meibomian gland. Histologic analysis of human, primate, steer, and rabbit glands revealed evidence of keratinized epithelium extending throughout the meibomian gland duct. Characteristic ultrastructural features of keratinized epithelium identified in primate and rabbit glands included tonofilaments, keratohyaline granules, lamellar bodies, and keratinized squamous cells. Comparison of the meibomian gland duct to the pilosebaceous canal and the sebaceous duct brought out certain dissimilarities such as (1) the lack of a well-developed stratum granulosum and (2) the absence of lipid inclusions within transitional cells from duct to acini. We postulate that abnormalities of the keratinizing process may be responsible for meibomian gland dysfunction states.

Meibomian gland dysfunction. II. The role of keratinization in a rabbit model of MGD.

Meibomian gland dysfunction (MGD) was induced in 34 albino rabbits by the twice-daily topical application of 2% epinephrine over a period of 6 months to 1 year. Seven age-matched control rabbits, not receiving epinephrine, were followed up for a similar period. All lids were evaluated pre- and post-treatment by gross clinical examination and by transillumination biomicroscopy and photography. Of the 68 rabbit lids evaluated, 56% developed signs of MGD, which ranged from plugging of the meibomian gland orifice and presence of microcysts (subclinical lesions; 30.9% of the lids) to opacification and enlargement of the glands with increasing severity (clinical lesions; 25.0% of the lids). The remaining lids (44%) remained normal. MGD did not develop in the seven control rabbits. After the development of MGD, lids were evaluated by immunofluorescent microscopy, SDS-PAGE and Western blotting using mouse monoclonal antibodies to keratin proteins. Development and progression of MGD in the rabbit appears to correlate with increasing stratification and keratinization of the meibomian gland duct epithelium. In the early stages of MGD, focal areas of epithelial hyperkeratinization were identified by immunohistochemical staining using AE2 monoclonal antibody, specific for the 56.5 kD and 65-67 kD keratin protein marker for keratinized epidermis. As the severity of MGD progressed there was progressive increase in the AE2 staining of the duct epithelium. SDS-PAGE and immunoblotting of proteins from meibomian gland excreta in chronic MGD showed a progressive increase in both the 56.5 kD and 65-67 kD keratinization protein markers during development of MGD. We conclude that hyperkeratinization of the duct epithelium leading to plugging and dilation of the meibomian gland underlies the development of MGD following topical epinephrine treatment.

Meibomian gland dysfunction. III. Meibomian gland lipids.

The lipid components of meibomian gland excreta were evaluated in rabbits after they received 2% topical epinephrine dropped into their eyes daily for a period of 6 months to a year to induce meibomian gland dysfunction (MGD). Changes were compared to excreta obtained from seven age-matched, untreated control rabbits. Comparison of the lipids from MGD lids with lipids obtained from control rabbits revealed, for clinically evident MGD, a marked increase in the lipids that are uniquely characteristic of epidermal tissue. These epidermal lipids are free sterols (large amounts) and a group of seven types of ceramides. The data are consistent with the hypothesis that the initiating factor of rabbit MGD is hyperkeratinization of the ductal epithelia. For clinically apparent MGD some hydrolysis of the sterol esters of the meibomian gland lipids also seems to have occurred. This was evidenced by the formation of an 8-fold increase in a cluster of anteiso fatty acids with chain lengths longer than C20 in the free fatty acid fraction. This group of free fatty acids was the same as the acids esterified to the sterol esters. We could detect no change in the lipid excreta obtained from rabbits that developed only subclinical MGD, consisting of orifice plugging and dilation of the duct.

The lipids of chalazia.

The tissue contents of chalazia taken from five individuals were extracted for lipids and analyzed by TLC and GLC. Lipids found were sterol esters 9%, wax esters 0%, triacyl glycerols 4%, free cholesterol 11%, free fatty acids 2%, unidentified nonpolar material 3%, ceramides 7%, cerebrosides 3%, phosphatidyl ethanolamine 12%, phosphatidyl choline 20%, sphingomyelin 4%, lysolecithin 4%, unidentified polar material 18.0%. GLC of the sterol esters gave peaks corresponding to the C14, C16, C18 and C20 normal fatty acid esters of cholesterol. The free fatty acids were also of the normal kind. Thus, the lipids of chalazia contents show little similarity to the lipids of the meibomian gland. The latter has a large component of sterol esters and wax esters. Also the fatty acids of all the meibomian gland ester lipids have significant amounts of iso- and anteisobranching in contrast to the chalazia lipids. Chalazia lipids more closely resemble the lipids of membranes of phagocytic cells with a large cholesterol content.