The basic functions of phosphoglycerate kinase 1 and its roles in cancer and other diseases.

Phosphoglycerate kinase 1 (PGK1) is an essential enzyme that catalyzes adenosine 5'-triphosphate (ATP) production in aerobic glycolysis. In addition to regulating cell metabolism, PGK1 is involved in multiple biological activities, including angiogenesis, mediated autophagy starting, binding of plasminogen, the DNA replication and repair, the proliferation and metastasis of tumor cells, cell invasion (a part of the flagellar axoneme and viral replication and it occurs mainly in protists), and is also associated with resistance to chemotherapy and prognosis of cancer patients. In this review, we focus on the basic functions of PGK1 and the relationship between PGK1 and different diseases, indicating that PGK1 has a broad application prospect to find a potential biomarker for tumor prognosis and an effective inhibitor.

Evidence for loss of a partial flagellar glycolytic pathway during trypanosomatid evolution.

Classically viewed as a cytosolic pathway, glycolysis is increasingly recognized as a metabolic pathway exhibiting surprisingly wide-ranging variations in compartmentalization within eukaryotic cells. Trypanosomatid parasites provide an extreme view of glycolytic enzyme compartmentalization as several glycolytic enzymes are found exclusively in peroxisomes. Here, we characterize Trypanosoma brucei flagellar proteins resembling glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK): we show the latter associates with the axoneme and the former is a novel paraflagellar rod component. The paraflagellar rod is an essential extra-axonemal structure in trypanosomes and related protists, providing a platform into which metabolic activities can be built. Yet, bioinformatics interrogation and structural modelling indicate neither the trypanosome PGK-like nor the GAPDH-like protein is catalytically active. Orthologs are present in a free-living ancestor of the trypanosomatids, Bodo saltans: the PGK-like protein from B. saltans also lacks key catalytic residues, but its GAPDH-like protein is predicted to be catalytically competent. We discuss the likelihood that the trypanosome GAPDH-like and PGK-like proteins constitute molecular evidence for evolutionary loss of a flagellar glycolytic pathway, either as a consequence of niche adaptation or the re-localization of glycolytic enzymes to peroxisomes and the extensive changes to glycolytic flux regulation that accompanied this re-localization. Evidence indicating loss of localized ATP provision via glycolytic enzymes therefore provides a novel contribution to an emerging theme of hidden diversity with respect to compartmentalization of the ubiquitous glycolytic pathway in eukaryotes. A possibility that trypanosome GAPDH-like protein additionally represents a degenerate example of a moonlighting protein is also discussed.

Spermatogenesis associated retrogenes are expressed in the human ovary and ovarian cancers.

BACKGROUND: Ovarian cancer is the second most prevalent gynecologic cancer in women. However, it is by far the most lethal. This is generally attributed to the absence of easily detectable markers specific to ovarian cancers that can be used for early diagnosis and specific therapeutic targets. METHODOLOGY/PRINCIPAL FINDINGS: Using end point PCR we have found that a family of retrogenes, previously thought to be expressed only in the male testis during spermatogenesis in man, are also expressed in normal ovarian tissue and a large percentage of ovarian cancers. In man there are at least eleven such autosomal retrogenes, which are intronless copies of genes on the X chromosome, essential for normal spermatogenesis and expressed specifically in the human testis. We tested for the expression of five of the known retrogenes, UTP14C, PGK2, RPL10L, RPL39L and UBL4B in normal human ovary and ovarian cancers. CONCLUSIONS/SIGNIFICANCE: We propose that the activation of the testis specific retrogenes in the ovary and ovarian cancers is of biological significance in humans. Because these retrogenes are specifically expressed in the ovary and ovarian cancers in the female they may prove useful in developing new diagnostic and/or therapeutic targets for ovarian cancer.

Glycolitic enzymes are targets of oxidation in aged human frontal cortex and oxidative damage of these proteins is increased in progressive supranuclear palsy.

Progressive supranuclear palsy (PSP) is a neurodegenerative disorder pathologically characterized by neuronal loss and gliosis mainly in specific subcortical nuclei, but also in the cerebral cortex. In addition to neuron loss, hyperphosphorylated tau deposition is found in neurons, astrocytes and coiled bodies. Limited studies have shown that certain oxidative products are increased in the PSP brain. The present study examines oxidative damage in the frontal cortex in 7 PSP compared with 8 age-matched controls. Western blotting of the frontal cortex showed increased 4-hydroxy-2-nonenal (HNE)-immunoreactive bands between 40 and 50 kDa in PSP cases. Bi-dimensional gel electrophoresis and Western blotting, together with mass spectometry, were used to identify HNE-modified proteins. Oxidized phosphoglycerate kinase 1 (PGK-1) and fructose bisphosphate aldolase A (aldolase A) were identified in all cases and 4 of 7 PSP cases, respectively. In contrast, PGK-1 and aldolase A were oxidized in 3 of 8 controls. Immunohistochemistry revealed the localization of aldolase A in neurons and astrocytes, and PGK-1 mainly in astrocytes. These findings show that PGK-1 and aldolase A are targets of oxidation in the frontal cortex in the aged human cerebral cortex and that oxidative damage of these proteins is markedly increased in the frontal cortex in PSP.

Analysis of enzymopathies in the human red blood cells by constraint-based stoichiometric modeling approaches.

The human red blood cell (RBC) metabolism is investigated by calculating steady state fluxes using constraint-based stoichiometric modeling approaches. For the normal RBC metabolism, flux balance analysis (FBA) is performed via optimization of various alternative objective functions, and the maximization of production of ATP and NADPH is found to be the primary objective of the RBC metabolism. FBA and two novel approaches, minimization of metabolic adjustment (MOMA) and regulatory on-off minimization (ROOM), which can describe the behavior of the metabolic networks in case of enzymopathies, are applied to observe the relative changes in the flux distribution of the deficient network. The deficiencies in several enzymes in RBC metabolism are investigated and the flux distributions are compared with the non-deficient FBA distribution to elucidate the metabolic changes in response to enzymopathies. It is found that the metabolism is mostly affected by the glucose-6-phosphate dehydrogenase (G6PDH) and phosphoglycerate kinase (PGK) enzymopathies, whereas the effects of the deficiency in DPGM on the metabolism are negligible. These stoichiometric modeling results are found to be in accordance with the experimental findings in the literature related to metabolic behavior of the human red blood cells, showing that human RBC metabolism can be modeled stoichiometrically.

Identification of immunodominant autoantigens in rat autoimmune orchitis.

Infection and inflammation of the genital tract are amongst the leading causes of male infertility. Experimental autoimmune orchitis (EAO) in the rat serves as a model for the investigation of inflammatory testicular impairment. In this study, experiments were conducted to identify the molecules that are responsible for eliciting the autoimmune attack on the testis. EAO was induced in in-bred Wistar rats by active immunization with testis homogenates (EAO group I). Development of disease was observed using histological techniques and a new non-invasive three-dimensional (3D) imaging technology for in vivo monitoring, termed flat-panel volumetric computed tomography (fpvCT). Examination of control and EAO testes demonstrated the superior image quality of high-resolution fpvCT. A proteomics approach using 2D SDS-PAGE and immunoblotting analysis with EAO sera identified 12 spots. Seven were subsequently identified by mass spectrometry as heat shock proteins 60 (Hsp60) and 70 (Hsp70), disulphide isomerase ER-60, alpha-1-anti-trypsin, heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1), sperm outer dense fibre major protein 2 (ODF-2), and phosphoglycerate kinase 1. Hsp70, ODF-2, hnRNP H1, and ER-60 were identified by all EAO sera studied. To test the capacity of the identified proteins to elicit testicular autoimmune disease, recombinant proteins were used either individually or in combination to immunize rats (EAO group II). In all groups, the incidence of EAO was 25%. Inflammatory-type (ED1+) and resident (ED2+) macrophages, lymphocytes (CD45RA+), and dendritic cells (Ox-62+) were strongly increased in EAO group II animals, comparable to the testes of EAO I rats. Pre-immunization with a low dose of recombinant Hsp 70, hnRNP H1 or ODF-2 before induction of EAO with testis homogenate significantly delayed the onset of EAO but could not prevent disease. The identification of testicular autoantigens will allow a better understanding of disease pathogenesis and could provide a basis for the development of novel therapies for inflammation-based male infertility.

Retrovirus silencing, variegation, extinction, and memory are controlled by a dynamic interplay of multiple epigenetic modifications.

Retrovirus silencing in stem cells produces silent or variegated provirus. Additional memory and extinction mechanisms act during differentiation. Here we show that retrovirus is silent or variegated in mouse embryonic stem (ES) cells that are de novo methyltransferase (dnmt3a and dnmt3b) null. Memory is maintained during differentiation, and extinction occurs on variegated retrovirus, indicating that DNA methylation is dispensable for all forms of retrovirus silencing. Silent and variegated provirus are marked by hypoacetylated histone H3 and bound H1. In wild-type ES cells, silent and variegated proviruses are methylated and bound by hypoacetylated H3, MeCP2, and less H1. Silencing, variegation, and extinction are partially reactivated by 5-AzaC in this context. Lentivirus vectors are also silent or variegated, marked by silent chromatin, and exhibit memory and extinction. We conclude that the universal epigenetic mark of retrovirus silencing is silent chromatin established via the dynamic interplay of multiple epigenetic modifications that include but do not require DNA methylation. A molecular mechanism of competitive H1 and MeCP2 binding may account for this epigenetic interplay, and a model for variegation is discussed.

Direct quantification of mRNA expression levels using single molecule detection.

Determination of the gene expression by direct quantification of mRNA is becoming increasingly important in basic, pharmaceutical and clinical research. We present a novel approach for gene quantification based on direct hybridization of gene-specific probes to target mRNA sequences in solution at temperatures ensuring absolute specificity of the probe-target complex. No enzymatic steps like reverse transcription or amplification by PCR are involved within the quantification process. In order to increase specificity as well as sensitivity, two probes emitting fluorescence light in different colors are used for our homogeneous assay using fluorescence cross-correlation. We relate to the single molecule sensitivity of excitation and detection in confocal cavities avoiding the amplification of the detected signal. The analysis of the expression level of high, medium and low abundant genes is described in two different cell lines, whereby the genes are quantified in absolute numbers.

A quantitative method for assessing co-localization in immunolabeled thin section electron micrographs.

A method is introduced for the analysis of nearest neighbor distances between immunogold particles marking proteins on electron micrographs. Deviation from the distribution that is predicted by chance indicates co-localization of the labeled species, and the potential for productive interaction in vivo. Application of this method to the analysis of nearest neighbor distances in experiments with pea leaf thin sections and isozyme-directed antibodies indicates that glyceraldehyde-3-P dehydrogenase is located near P-glycerate kinase and near aldolase in the chloroplast stroma, consistent with the notion that these enzymes are part of a multi-enzyme photosynthetic CO(2)-fixation complex in situ.

Ultrastructural localization of phosphoglycerate kinase in adult Clonorchis sinensis.

Phosphoglycerate kinase (PGK) is an enzyme that produces one ATP molecule in the glycolytic pathway. Clonorchis sinensis is largely dependent on glycolysis for energy production. We performed immunoelectron microscopy on adult C. sinensis by using mouse immune serum raised against recombinant C. sinensis PGK. A high density of gold particles was found in the microvilli of the intestinal epithelium and in lamellae of the sperm duct. PGK was common in the somatic cells of intra-uterine eggs and in excreted products. It was localized with moderate intensity in muscular fibers of the subtegumental muscle layer, and in the myoepithelia of the intestine and excretory bladder. We suggest that PGK plays an essential role in C. sinensis energy production for movement via muscle contraction.

Colliding primary lung cancers of adenosquamous carcinoma and large cell neuroendocrine carcinoma.

We report an extremely rare case of primary lung cancer showing various histological elements diagnosed as the collision of an adenosquamous carcinoma and a large cell neuroendocrine carcinoma by loss of heterozygosity (LOH) analysis of the human androgen receptor (AR) and phosphoglycerate kinase (PGK-1) genes. The tumor exhibited a tiny ground-glass opaque shadow suggesting atypical adenomatous hyperplasia 18 months prior to surgery. However, the tumor grew rapidly, and the resected tumor consisted of two closely located nodules. The larger nodule was composed of well-differentiated adenocarcinomatous and moderately to poorly differentiated squamous cell carcinomatous elements, while the smaller nodule consisted of a large cell neuroendocrine carcinomatous element with partial squamoid differentiation having focal continuity with the adenocarcinomatous element. Both the adenocarcinomatous and squamous cell carcinomatous elements revealed transitional features and LOH of AR and PGK-1 genes, while the large cell neuroendocrine carcinomatous element showed a monoclonal pattern but possessed both alleles of AR and PGK-1 genes. From these clinical and pathological results, the parental cell of the large cell neuroendocrine carcinomatous element was considered to be different from that of the adenosquamous carcinomatous element.

Induction of heat shock proteins DnaK, GroEL, and GroES by salt stress in Lactococcus lactis.

The bacterium Lactococcus lactis has become a model organism in studies of growth physiology and membrane transport, as a result of its simple fermentative metabolism. It is also used as a model for studying the importance of specific genes and functions during life in excess nutrients, by comparison of prototrophic wild-type strains and auxotrophic domesticated (dairy) strains. In a study of the capacity of domesticated strains to perform directed responses toward various stress conditions, we have analyzed the heat and salt stress response in the established L. lactis subsp. cremoris laboratory strain MG1363, which was originally derived from a dairy strain. After two-dimensional separation of proteins, the DnaK, GroEL, and GroES heat shock proteins, the HrcA (Orf1) heat shock repressor, and the glycolytic enzymes pyruvate kinase, glyceral-dehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase were identified by a combination of Western blotting and direct N-terminal amino acid sequencing of proteins from the gels. Of 400 to 500 visible proteins, 17 were induced more than twofold during heat stress. Two classes of heat stress proteins were identified from their temporal induction pattern. The fast-induced proteins (including DnaK) showed an abruptly increased rate of synthesis during the first 10 min, declining to intermediate levels after 15 min. GroEL and GroES, which also belong to this group, maintained a high rate of synthesis after 15 min. The class of slowly induced proteins exhibited a gradual increase in the rate of synthesis after the onset of stress. Unlike other organisms, all salt stress-induced proteins in L. lactis were also subjected to heat stress induction. DnaK, GroEL, and GroES showed similar temporal patterns of induction during salt stress, resembling the timing during heat stress although at a lower induction level. These data indicate an overlap between the heat shock and salt stress responses in L. lactis.

Trypanosoma brucei: identification of an internal region of phosphoglycerate kinase required for targeting to glycosomal microbodies.

Among the microbodies found in eukaryotes are the glycosomes of Trypanosoma brucei, thought to be closely related to peroxisomes. Two types of targeting signals for glycosomes have been identified thus far: type 1 at the C-terminus and type 2 at the N-terminus. In this report, we use an epitope-tagging system to characterize the targeting signal found on the minor glycosomal isozyme of phosphoglycerate kinase, 56PGK. No type 1 or 2 signal was found; rather, the topogenic information was found to be internal. Chimeric molecules formed with the cytoplasmic phosphoglycerate kinase isozyme indicate that a region between amino acids 24 and 91 of 56PGK is essential for glycosomal targeting. No homology was found between this region and peroxisomal proteins containing internal targeting signals.

Organization, sequence and stage-specific expression of the phosphoglycerate kinase genes of Leishmania mexicana mexicana.

In Leishmania mexicana two genes were detected coding for different isoforms of the glycolytic enzyme phosphoglycerate kinase. This situation contrasts with that observed in other Trypanosomatidae (Trypanosoma brucei, Trypanosoma congolense, Crithidia fasciculata) analyzed previously, which all contain three different genes coding for isoenzymes A, B and C, respectively. All attempts to detect in L. mexicana a type A PGK, or a gene encoding it, proved unsuccesful. We have cloned and characterized the genes PGKB and PGKC. They code for polypeptides of 416 and 478 amino acids with a molecular mass of 45146 and 51318 Da, respectively. The two polypeptides are 99% identical. PGKC is characterized by a 62 residue C-terminal extension with alternating stretches of hydrophobic and charged, mainly positive amino acids. As in other Trypanosomatidae, PGKB is located in the cytosol, PGKC in the glycosomes. However, Leishmania mexicana distinguishes itself from other trypanosomatids by the simultaneous expression of these isoenzymes: approximately 80% of PGK activity is found in the cytosol and 20% in the glycosomes, both in promastigotes and in the amastigote-like form of the parasite.

The majority of T lymphocytes are polyclonal during the chronic phase of chronic myelogenous leukemia.

To clarify the extent of cell lineage involvement in chronic myelogenous leukemia (CML), we investigated the bcr gene rearrangement and clonality using the X-chromosome-linked restriction fragment length polymorphism (RFLP) methylation method in T lymphocytes and granulocytes. We examined the granulocyte and T-cell fractions from the peripheral blood of seven female patients with CML during the chronic phase; patients were heterozygous for RFLPs at the phosphoglycerate kinase (PGK) or the hypoxanthine phosphoribosyltransferase (HPRT) gene. RFLP-methylation analysis of granulocytes demonstrated a monoclonal pattern in six of the seven patients and a rearranged bcr gene in all seven patients. In contrast, T lymphocytes exhibited a polyclonal pattern in six cases; in one case, a faint band was observed following methyl-sensitive enzyme cleavage. The bcr gene analysis in T lymphocytes showed the germline in every case. Our results indicate that the majority of T lymphocytes are polyclonal during the chronic phase of CML and confirm previous reports based on glucose-6-phosphate dehydrogenase, cytogenetic, and bcr rearrangement analyses.

Analysis of the cDNA and encoded protein of the human testis-specific PGK-2 gene.

Because of their unique function, germ cells require unique gene products. Thus, although the glycolytic enzyme phosphoglycerate kinase (PGK) is required in all metabolically active cell types, there are two functional PGK genes in the mammalian genome, one, PGK-1, that is X-linked and ubiquitously expressed in all somatic tissues, and a second, PGK-2, that is autosomal and expressed only in spermatogenic cells. Expression of the PGK-2 gene may function solely to compensate for repressed expression of the PGK-1 gene due to X-chromosome inactivation in spermatocytes. Alternative y, the PGK-2 gene could encode an isozyme with unique characteristics that are beneficial to spermatozoa. We have isolated a cDNA of the human PGK-2 gene and used this as probe to demonstrate that transcription of this gene in spermatocytes and spermatids coincides with a period of repressed transcription of the X-linked PGK-1 gene during spermatogenesis in the human testis. We have also analyzed the amino acid sequence and protein characteristics of the PGK-2 isozyme deduced from this cDNA and compared them with that of the human PGK-1 isozyme to show that known structural and functional motifs are conserved in both proteins. Finally, we have examined the distribution of the PGK-1 and PGK-2 isozymes during spermatogenesis in the mouse to show that while the PGK-2 protein does not appear to possess any unique intracellular localization signal, it is more stable in vivo than the PGK-1 protein.

Growth retardation and hyperglycemia in insulin-like growth factor binding protein-1 transgenic mice.

Transgenic mice that constitutively overexpressed rat insulin-like growth factor binding protein-1 (IGFBP-1) were generated to determine the effects of overexpression of IGFBP-1 on growth and development. In offspring of three of the founders that showed high levels of expression, birth weight was significantly reduced to approximately 83-92% of the weight of their nontransgenic littermates. The transgenic mice gained less weight and were approximately 3.5-8 g lighter than nontransgenic littermates at 40 days of age. The difference in body weight between transgenic and wild-type mice was most apparent around the time of weaning when transgenic mice showed a more marked growth deceleration than wild-type mice. No significant catch-up growth was apparent over the first 3 months of life. In addition, offspring of all three high-expressing founders demonstrated fasting hyperglycemia. The transgene was highly expressed in the brain, uterus, lung, kidney, and heart, but little expression was detected in the liver. The weight of the brain relative to body weight was significantly reduced in transgenic mice compared with wild-type mice, whereas the relative weight of most other organs was similar to wild-type mice. These data demonstrate that IGFBP-1 may function to inhibit IGF action in vivo and that this inhibition selectively impairs development of organs such as the brain.

Quantitative non-radioactive clonality analysis of human leukemic cells and progenitors using the human androgen receptor (AR) gene.

Clonal analysis of FACS-purified primitive hematopoietic stem cells and of their progeny as assessed by the progenitors obtained from long-term cultures requires PCR-based approaches, mainly because of the low number of cells available. We have developed a non-radioactive androgen receptor (AR) assay which allows a simple and quantitative evaluation of the clonality of hematopoietic cells and progenitors. In this approach 5' AR primer is labelled by fluorescein and the amplified product is run on a sequencing gel which allows evaluation of the intensity of the fluorescent peaks generated. A computer software then analyzes the reduction of the intensity of the peaks on HpaII-digested samples. In order to determine the feasibility of the technique, we analyzed the clonality of leukemic cells from a patient with an acute-phase CMML which showed a typical clonal pattern of her leukemic DNA sample (WBC = 300 x 10(9)/I) using phosphoglycerate kinase (PGK) analysis. The same sample was then analyzed with either radioactive- or fluorescein-labelled AR primers, showing a typical clonal pattern (complete disappearance of one allele after HpaII digestion). A short-term clonogenic assay was then set up on methylcellulose and clonogenic progenitors were individually analyzed. All 24 colonies tested showed a typical clonal pattern with the disappearance of the same allele on each sample after HpaII digestion, indicating that they all derived from the same leukemic stem cell. Using this approach we then analyzed 94 patients with several hematologic malignancies and quantification of their fluorescent peaks. Fifty-four percent of the patients were clearly heterozygous (ie, a difference of > or = 2 CAG repeats was present between the two copies of the gene) and could be analyzed in an automatic sequencer using the fluorescent primers. Bone marrow mononuclear cells from all patients with acute myeloid leukemia (AML) showed a clonal or oligoclonal pattern at diagnosis whereas a polyclonal pattern was seen when remission was obtained. Similarly, out of 21 patients with a diagnosis of myelodysplastic syndrome (MDS), a clonal pattern was demonstrated in 10 whereas an oligoclonal or non-clonal pattern was shown in 11. These results show that this non-radioactive and safe technology can now be used on a large scale to evaluate the clonality of highly purified hematopoietic stem cells and their progenitors in hematopoietic malignancies and this might allow new insights into the targets of clonal amplification.

Analysis of meningiomas by methylation- and transcription-based clonality assays.

The clonal derivation of tumors can be determined by X chromosome inactivation analysis based on differential expression of genes or differential methylation of cytosine residues in CpG islands near polymorphic loci. In this report, we compared a transcription-based RNA analysis with a methylation-based DNA assay to determine clonality of meningiomas. Both clonality assays use PCR-based analysis at the hunan androgen-receptor gene (HUMARA) on the X chromosome. Among 23 meningiomas from female patients, 19 were informative heterozygotes at this locus (83%). The patterns of X chromosome inactivation in four patients were extremely skewed towards one allele in blood (unequal Lyonization), which precluded clonality determination in the tumor samples. Concordant clonality results with methylation- and transcription-based clonality assays were demonstrated in 9 of 13 informative tumors expressing the androgen receptor. Seven meningiomas were monoclonal, but surprisingly, two pathologically documented cases of meningiomas were polyclonal. There was disparity in 4 of 13 tumor specimens that were polyclonal by the methylation-based assay but monoclonal by the transcription assay. Clonality examination of these tumors by the methylation-based phosphoglycerate kinase assay provided identical results to the methylation-based analysis at the HUMARA locus. In addition, loss of heterozygosity (LOH) studies of chromosome 22, which is frequently deleted in meningiomas, showed that four of four informative samples of the six polyclonal tumors had partial LOH in tumor tissues. However, complete LOH was observed in primary cultured cells, which were also monoclonal by the methylation assay. Taken together, these data suggest that the disparity of the two assays in these four cases may be due to differences in the level of expression of the androgen receptor gene in tumors. Therefore, we conclude that: (a) clonal derivation of meningiomas determined by both transcription- and methylation-based clonality assays are in full agreement in many (9 of 13) but not all cases (4 of 13); and (b) most meningiomas (9 of 15) are monoclonal in origin, whereas some meningioma samples (6 of 15) are polyclonal or may contain heterogeneous components.

Analysis of clonality by polymerase chain reaction for phosphoglycerate kinase-1. Heteroduplex generator.

Polymerase chain reaction (PCR) amplification has been used to determine the clonal composition of tissues based on analysis of the pattern of X-chromosome inactivation, but its use has been limited by technical difficulties. This report presents an expedited method to use PCR in the analysis of clonality. The method uses gel electrophoresis of heteroduplexes formed with an artificial heteroduplex generator (HG) and PCR products from the phosphoglycerate kinase-1 (PGK-1) gene from the tissue sections. Amplification was successful in 36 of 37 cases originally diagnosed as endometrial adenocarcinoma. HG analysis of 36 cases confirmed heterozygosity in 12 cases (33.3%). PGK-1 PCR amplification product was obtained from both control and lesional tissue in 10 of the 12 heterozygous cases. Of these 10 cases, seven were shown to consist of clonal cell populations by HG analysis. Two of three cases diagnosed as well-differentiated endometrioid adenocarcinoma were found to be comprised of polyclonal populations of cells. One case produced an anomalous pattern with HG analysis and was shown to be aneuploid by fluorescence in situ hybridization (FISH) with a chromosome X alpha-satellite probe. It is concluded that HG is a useful alternative to restriction fragment length polymorphism (RFLP) analysis of X-chromosome inactivation as a marker of tissue clonality in cases in women.