Mohinder Sarna

A novel ADP-ribosylation like factor (ARL-6), interacts with the protein-conducting channel SEC61β subunit

We report here the isolation of a new member of the ADP‐ribosylation factor (ARF)‐like family (ARL‐6) present in the J2E erythroleukemic cell line, but not its myeloid variants. Consistent with this lineage‐restricted expression, ARL‐6 mRNA increased with erythropoietin‐induced maturation of J2E cells, and decreased with interleukin 6‐induced differentiation of M1 monoblastoid cells. In tissues, ARL‐6 mRNA was most abundant in brain and kidney. While ARL‐6 protein was predominantly cytosolic, its membrane association increased following exposure to GTP‐γS, like many members of the ARF/ARL family. Using the yeast two‐hybrid system, six molecules which interact with ARL‐6 were identified including SEC61β, a subunit of the heterotrimeric protein conducting channel SEC61p. Co‐immunoprecipitation of ARL‐6 confirmed a stable association between ARL‐6 and SEC61β in COS cells. These results demonstrate that ARL‐6, a novel member of the ADP‐ribosylation factor‐like family, interacts with the SEC61β subunit.

Differential regulation of SOCS genes in normal and transformed erythroid cells

The SOCS family of genes are negative regulators of cytokine signalling with SOCS-1 displaying tumor suppressor activity. SOCS-1, CIS and SOCS-3 have been implicated in the regulation of red blood cell production. In this study, a detailed examination was conducted on the expression patterns of these three SOCS family members in normal erythroid progenitors and a panel of erythroleukemic cell lines. Unexpectedly, differences in SOCS gene expression were observed during maturation of normal red cell progenitors, viz changes to CIS were inversely related to the alterations of SOCS-1 and SOCS-3. Similarly, these SOCS genes were differentially expressed in transformed erythoid cells – erythroleukemic cells immortalized at an immature stage of differentiation expressed SOCS-1 and SOCS-3 mRNA constitutively, whereas in more mature cell lines SOCS-1 and CIS were induced only after exposure to erythropoietin (Epo). Significantly, when ectopic expression of the tyrosine kinase Lyn was used to promote differentiation of immature cell lines, constitutive expression of SOCS-1 and SOCS-3 was completely suppressed. Modulation of intracellular signalling via mutated Epo receptors in mature erythroleukemic lines also highlighted different responses by the three SOCS family members. Close scrutiny of SOCS-1 revealed that, despite large increases in mRNA levels, the activity of the promoter did not alter after erythropoietin stimulation; in addition, erythroid cells from SOCS-1−/− mice displayed increased sensitivity to Epo. These observations indicate complex, stage-specific regulation of SOCS genes during normal erythroid maturation and in erythroleukemic cells.

Transcription Factor Erythroid Krüppel-like Factor (ELKF) Is Essential for the Erythropoietin-induced Hemoglobin Production but Not for Proliferation, Viability, or Morphological Maturation

The erythroid Krüppel-like factor (EKLF) is essential for the transcription of βmaj globin in erythroid cells. We show here that RNA for this transcription factor did not alter during erythropoietin-induced differentiation of J2E cells; however, EKLF protein content decreased and was inversely related to globin production. This unexpected result was also observed during chemically induced maturation of two murine erythroleukemia cell lines. To explore the role of EKLF in erythroid terminal differentiation, an antisense EKLF construct was introduced into J2E cells. As a consequence EKLF RNA and protein levels fell by approximately 80%, and the cells were unable to manufacture hemoglobin in response to erythropoietin. The failure to produce hemoglobin was due to reduced transcription of not only globin genes but also key heme enzyme genes. However, numerous other genes, including several erythroid transcription factors, were unaffected by the decrease in EKLF. Although hemoglobin synthesis was severely impaired with depleted EKLF levels, morphological maturation in response to erythropoietin continued normally. Moreover, erythropoietin-induced proliferation and viability were unaffected by the decrease in EKLF levels. We conclude that EKLF affects a specific set of genes, which regulates hemoglobin production and has no obvious effect on morphological changes, cell division, or viability in response to erythropoietin.

Thyroid hormone receptor-interacting protein 1 modulates cytokine and nuclear hormone signaling in erythroid cells.

Erythropoietin (Epo) and thyroid hormone (T3) are key molecules in the development of red blood cells. We have shown previously that the tyrosine kinase Lyn is involved in differentiation signals emanating from an activated erythropoietin receptor. Here we demonstrate that Lyn interacts with thyroid hormone receptor-interacting protein 1 (Trip-1), a transcriptional regulator associated with the T3 receptor, providing a link between the Epo and T3 signaling pathways. Trip-1 co-localized with Lyn and the T3 receptor α in the cytoplasm/plasma membrane of erythroid cells but translocated to discrete nuclear foci shortly after Epo-induced differentiation. Our data reveal that T3 stimulated the proliferation of immature erythroid cells, and inhibited maturation promoted by erythropoietin. Removal of T3 reduced cell division and enhanced terminal differentiation. This was accompanied by large increases in the cell cycle inhibitor p27Kip1 and by increasing expression of erythroid transcription factors GATA-1, EKLF, and NF-E2. Strikingly, a truncated Trip-1 inhibited both erythropoietin-induced maturation and T3-initiated cell division. This mutant Trip-1 acted in a dominant negative fashion by eliminating endogenous Lyn, elevating p27Kip1, and blocking T3 response elements. These data demonstrate that Trip-1 can simultaneously modulate responses involving both cytokine and nuclear receptors.

C-ERBB-2 AMPLIFICATION AND OVEREXPRESSION IN BREAST CANCER : EVALUATION AND COMPARISON OF SOUTHERN BLOT, SLOT BLOT, ELISA AND IMMUNOHISTOCHEMISTRY

&NA; The oncogene c‐erbB‐2 has been shown to be amplified in 17‐30% of breast cancers, with similar levels of overexpression of the oncogene product p185, a transmembrane growth factor receptor glycoprotein. Amplification of c‐erbB‐2 is now generally considered to be a significant prognostic indicator in patients with breast cancer. A series of 74 consecutive breast carcinomas were analysed for c‐erbB‐2 amplification and p185 overexpression. The procedures of Southern blotting and slot blot were used for the analysis of oncogene amplification, while immunoperoxidase (IPOX) staining and enzyme‐linked immunosorbent assay (ELISA) were used for the analysis of p185 overexpression. Detection of c‐erbB‐2 oncogene amplification by both the conventional Southern blotting technique and by the slot blot technique showed complete accord, with the amplified c‐erbB‐2 oncogene being detected in 14 of the 74 patients (18.9%). The c‐erbB‐2 oncoprotein, as measured by IPOX and ELISA, was found to be overexpressed in 21% and 19% of patients, respectively. Comparison was made between the results attained by all four methods, and further comparison of the techniques was made from the point of view of ease of use, expense and ease of introduction into routine diagnostic laboratories. Immunocytochemistry in combination with slot blotting procedures were considered to be the most cost effective methods for evaluation of overexpression and amplification in routine pathology laboratories.

Evaluation of the expression levels of nm23-h1 mRNA in primary breast cancer, benign breast disease, axillary lymph nodes and normal breast tissue

&NA; Expression levels of nm23‐H1 were evaluated in a variety of normal, benign and malignant breast tissues by Northern and slot blot. Tissues from 153 patients presenting with palpable breast lesions were studied: 132 primary infiltrating breast cancers, 9 pure duct carcinoma in situ lesions, a phyllodes tumor, 9 benign lesions and 2 local recurrences of carcinoma. In addition to lesional tissue, 49 samples of macroscopically normal breast tissue, 37 axillary lymph nodes and 9 samples from patients undergoing cosmetic reduction mammoplasty were studied. Sets of normal breast tissue, primary tumor and lymph node tissue from individual patients were available for comparison in 37 cases. A wide range of gene expression was detected in the various tissue types. The highest levels of expression were detected in malignant samples with in situ carcinomas being associated with the highest levels of gene expression. The expression levels of nm23‐H1 in normal breast tissue were lower than the corresponding tumors from the same patients (p < 0.0005). Benign breast lesions (including 6 fibroadenomas) had levels of gene expression approximating those of the normal tissue samples. Normal axillary lymph nodes had significantly lower levels of nm23‐H1 expression than nodes with metastatic deposits (p < 0.03). No significant association was observed between nm23‐H1 expression levels and axillary node status in patients with infiltrating carcinoma, although there was a slight trend toward lower nm23‐H1 mRNA levels in the node negative group. Some special tumor types known to metastasize infrequently (tubular, tubulo‐lobular carcinoma) had low levels of expression, whilst others (colloid carcinoma) exhibited high levels of expression. Comparison of samples of primary tumor, surrounding normal breast and metastatic node deposits in individual patients showed no clear trends in gene expression between these tissue types. The validity of nm23‐H1 as a marker of metastatic potential is questioned on the basis of these results. The higher levels of expression observed in malignant tissue suggest that nm23‐H1 may be involved in tumorigenesis, but clear evidence for this, and the mechanisms by which the gene may influence tumor biology, remain to be determined.

Dominant action of mutated erythropoietin receptors on differentiation in vitro and erythroleukemia development in vivo

J2E cells produce rapid, fatal erythroleukemias in vivo but still respond to erythropoietin (epo) in vitro by differentiating, proliferating and remaining viable in the absence of serum. Mutant epo receptors were introduced into these cells to determine whether they could influence the different biological responses to epo in vitro and the development of erythroleukemias. Three mutant receptors were used as cytoplasmic truncation mutants Δ257 and Δ321 (above box 1 and below box 2 respectively), and the cytoplasmic point mutant W282R (defective for JAK2 activation). Strikingly, the Δ321 mutation produced a hyper-sensitive response in vitro to epo-induced differentiation and viability, but not to proliferation. In contrast with the Δ321 receptor, the Δ257 and W282R mutants inhibited all biological responses to epo due to impaired JAK2 phosphorylation. Significantly, erythroleukemias took almost twice as long to develop with cells containing the W282R mutation, indicating that JAK2 plays an important role in the emergence of these leukemias. These data demonstrate that mutant epo receptors dominantly altered responses of J2E cells to epo in culture and the development of erythroleukemias.

nm23-H1 metastasis suppressor gene expression in primary breast cancer: associations with axillary lymph node status, tumour size, type and grade

Abstract The nm23 -H1 gene has tumour suppressor functions and is a putative metastasis suppressor. Higher levels of expression of the gene in breast carcinoma have been associated with the absence of lymph node metastasis, and with longer relapse free intervals and overall survival. Expression levels of nm23 -H1 in breast carcinoma were examined by Northern and slot blot in a consecutive series of 105 patients. The levels of nm23 -H1 mRNA expression were examined for associations with axillary lymph node metastasis, and with tumour size, tumour type and grade, mitotic rate, nuclear size and pleomorphism and vascular invastion, in 97 cases of invasive cancer. No statistically significant differences in nm23 expression levels between axillary node negative and axillary node positive infiltrating duct carcinomas were observed. There was no correlation between nm23 -H1 expression levels and other histopathologically determined prognostic factors. Higher levels of gene expression were demonstrated in pure ductal carcinoma in situ (DCIS) lesions when compared to invasive tumours; further analysis suggested that DCIS components within invasive tumours contributed to measurements of nm23 expression, possibly masking the lower expression levels of the invasive component in some cases. Further correlative studies, using in situ hybridization or gene product immunohistochemistry would be most suited to determining the contribution of different tissue components in breast cancer.