Overexpression Of Cyclin D1 mRNA Research Articles

Tissue-specific regulatory regions of the PTH gene localized by novel chromosome 11 rearrangement breakpoints in a parathyroid adenoma

Parathyroid adenomas can contain clonal rearrangements of chromosome 11 that activate the cyclin D1 oncogene through juxtaposition with the PTH gene. Here we describe such a chromosomal rearrangement whose novel features provide clues to locating elusive cis-regulatory elements in the PTH gene and also expand the physical spectrum of pathogenetic breakpoints in the cyclin D1 gene region. Southern blot analyses of the parathyroid adenoma revealed rearrangement in the PTH gene locus. Analysis of rearranged DNA clones that contained the breakpoint, obtained by screening a tumor genomic library, pinpointed the breakpoint in the PTH locus 3.3 kb upstream of the first exon. Accordingly, highly conserved distal elements of the PTH 5' regulatory region were rearranged at the breakpoint approximately 450 kb upstream of the cyclin D1 oncogene, resulting in overexpression of cyclin D1 mRNA. Thus, PTH-cyclin D1 gene rearrangement breakpoints in parathyroid tumors can be located far from those previously recognized. In addition to expanding the molecular spectrum of pathogenetic chromosomal lesions in this disease, features of this specific rearrangement reinforce the existence of one or more novel cis-enhancer/regulatory elements for PTH gene expression and narrow their location to a 1.7-kb DNA segment in the distal PTH promoter.

Phase III study of patients with relapsed, refractory mantle cell lymphoma treated with temsirolimus compared with investigator’s choice therapy

8513 Background: Temsirolimus (TEMSR) is a specific inhibitor of mTOR, a kinase that regulates translation of key cell cycle proteins such as cyclin D1. Mantle cell lymphoma (MCL) is characterized by an 11;14 translocation that results in overexpression of cyclin D1 mRNA. In this phase 3, randomized, open-label study, we compared the antitumor activity of TEMSR with an investigator’s choice of therapy (IC) in patients (pts) with relapsed and/or refractory MCL. Methods: To test the hypothesis that TEMSR would increase progression-free survival (PFS) compared with IC (assuming medians of 6.2 and 3.0 mo, respectively), accrual of 177 pts to achieve 105 PFS events was planned. Power was 80% and α=0.025 comparing TEMSR with IC. Pts must have received 2–7 prior lines of therapy and received an alkylating agent, an anthracycline, and rituximab. Pts were randomly assigned (1:1:1) to 1 of 2 schedules of IV TEMSR, 175 mg 3x weekly followed by either 75 mg (175/75, arm 1) or 25 mg (175/25, arm 2) weekly, or to IC (arm 3) with the single agents gemcitabine (42%); fludarabine (26%); chlorambucil, cladribine, etoposide (6% ea); cyclophosphamide, thalidomide, vinblastine (4% ea); or alemtuzumab, lenalinomide (2% ea). The primary endpoint was PFS based on central independent review of radiologic and clinical data. Results: We report final results for 162 pts (54 pts/arm, median age 67 y, 81% male, 50% >3 prior regimens, 32% prior stem cell transplant) after 105 PFS events. Treatment with TEMSR 175/75 mg resulted in significant improvement in PFS and objective response rate and a trend toward longer overall survival compared with IC (Table). The most frequently occurring adverse events ≥gr 3 were thrombocytopenia (arm 1: arm 2: arm 3, 59%: 52%: 36% pts), anemia (20%: 11%: 17%), neutropenia (15%: 22%: 26%), and asthenia (13%: 19%: 8%). Conclusions: With an acceptable safety profile, TEMSR 175/75 mg significantly increased PFS and objective response rate of pts with relapsed, refractory MCL compared with IC. Parameter TEMSR, 175/75 mg Arm 1 TEMSR, 175/25 mg Arm 2 IC, Arm 3 Progression-free survival, independent assessment Median (97.5% CI), mo 4.8 (3.1, 8.1) 3.4 (1.9, 5.5) 1.9 (1.6, 2.5) Increase in median * 153% 79% Hazard ratio (97.5% CI) * 0.44 (0.25, 0.78) 0.65 (0.39, 1.10) p-value * 0.0009 0.0618 Overall survival Median (95% CI), mo 10.9 (8.1, 14.1) 8.5 (5.8, 14.0) 5.8 (4.8, 12.4) Increase in median * 88% 47% Hazard ratio (95% CI) * 0.62 (0.37, 1.05) 0.80 (0.48, 1.33) p value * 0.0714 0.3876 Obj response rate (95% CI) 22% (11, 33) 6% (0, 12) 2% (0, 5) p-value * 0.0019 0.6179 * Arm 1 or arm 2 : arm 3 Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Wyeth Research Wyeth Wyeth Pharmaceuticals Millennium, Wyeth

Alteration of Cyclin D1 in Chinese Patients with Breast Carcinoma and its Correlation with Ki-67, pRb, and p53

For the female population in Asia, systematic investigation on alterations of cyclin D1 in breast carcinoma is rare, and correlation between cyclin D1 expression with clinicopathological parameters, survival rate, and other prognostic marker associated with cell cycle is unclear. Expression of cyclin D1 protein, Ki-67, pRb, and p53 was determined by immunohistochemistry in 18 cases of early breast carcinomas and 80 cases of invasive ductal carcinomas. Genetic alteration of cyclin D1 gene and overexpression of cyclin D1 mRNA were detected by Southern blot and RT-PCR, respectively. Expression of cyclin D1 is negative in usual ductal hyperplasia (UDH) and atypical ductal hyperplasia (ADH). However, in 52.0% (51/98) of all breast carcinomas, positive expression of cyclin D1 was observed. Five-year survival rate of the patients with positive expression of cyclin D1 (52.7%) is significantly lower than the cases with negative expression of cyclin D1 (72.1%). Positive rate of cyclin D1 protein in invasive ductal carcinoma (52.5%) is slightly higher than overexpression rate (40.8%) of cyclin D1 mRNA but significantly higher than amplification rate of cyclin D1 gene (18.4%). Expression of cyclin D1 is correlated with Ki-67 expression, but not correlated with pRb and p53 expression. Positive expression of cyclin D1 could serve as a poor prognostic marker for Chinese patients with breast carcinoma independent of nodal metastasis and clinical stage. Expression of cyclin D1 protein is affected more directly by overexpression of cyclin D1 mRNA rather than cyclin D1 gene amplification. The cooperation between pRb and p53 with cyclin D1 protein in the carcinogenesis of breast carcinoma is not supported by the results.

Detection of cyclin D1 mRNA by reverse transcription-polymerase chain reaction in paraffin-embedded tissues and its diagnostic significance for mantle cell lymphoma

To investigate the feasibility of detecting cyclin D1 mRNA in paraffin-embedded tissues by reverse transcriptase polymerase chain reaction (RT-PCR) and competitive RT-PCR and its diagnostic and differential diagnostic significance for mantle cell lymphoma (MCL). Paraffin-embedded samples of 36 cases of MCL, 71 cases of other small B-cell lymphomas and 20 cases of lymphoid reactive hyperplasia as control group were retrieved from archival materials. Cyclin D1 protein and its mRNA was detected by EnVision and RT-PCR and competitive RT-PCR in all samples. House-keeping gene PGK was choosen as internal control. (1) Cyclin D1 protein was expressed in 27 of the 38 MCL (71.1%). No cyclin D1 expression was found in the control group. (2) PGK was detected in 103 of the 116 cases (88.8%) and also detected in 34 of 36 MCL cases (94.7%). (3) cyclin D1 mRNA was detected in 34 nodal mantle cell lymphoma cases by RT-PCR in paraffin-embedded tissues. The positive rate of cyclin D1 mRNA was 94.4% in mantle cell lymphomas after exclusion of the 2 cases which were negative for both cyclin D1 mRNA and PGK. cyclin D1 mRNA was not detected in other nodal small B-cell lymphomas or lymphoid reactive hyperplasia, except 1 case of B-SLL. Sequencing analysis showed that sequences were identical to cyclin D1. (4) Cyclin D1 mRNA overexpression was detected in 27 cases of nodal mantle cell lymphoma by competitive RT-PCR in paraffin-embedded tissues. The positive rate of cyclin D1 mRNA overexpression was 75.0% in mantle cell lymphomas after exclusion of 2 cases which were negative for both cyclin D1 mRNA and PGK. cyclin D1 mRNA overexpression was not detected in other nodal small B-cell lymphomas or lymphoid reactive hyperplasia. RT-PCR and competitive RT-PCR detection of cyclin D1 mRNA overexpression could be used for the diagnosis and differential diagnosis of mantle cell lymphoma in paraffin-embedded blocks.

An Unliganded Thyroid Hormone β Receptor Activates the Cyclin D1/Cyclin-Dependent Kinase/Retinoblastoma/E2F Pathway and Induces Pituitary Tumorigenesis

Thyroid-stimulating hormone (TSH)-secreting tumors (TSH-omas) are pituitary tumors that constitutively secrete TSH. The molecular genetics underlying this abnormality are not known. We discovered that a knock-in mouse harboring a mutated thyroid hormone receptor (TR) beta (PV; TRbeta(PV/PV) mouse) spontaneously developed TSH-omas. TRbeta(PV/PV) mice lost the negative feedback regulation with highly elevated TSH levels associated with increased thyroid hormone levels (3,3',5-triiodo-l-thyronine [T3]). Remarkably, we found that mice deficient in all TRs (TRalpha1(-/-) TRbeta(-/-)) had similarly increased T3 and TSH levels, but no discernible TSH-omas, indicating that the dysregulation of the pituitary-thyroid axis alone is not sufficient to induce TSH-omas. Comparison of gene expression profiles by cDNA microarrays identified overexpression of cyclin D1 mRNA in TRbeta(PV/PV) but not in TRalpha1(-/-) TRbeta(-/-) mice. Overexpression of cyclin D1 protein led to activation of the cyclin D1/cyclin-dependent kinase/retinoblastoma protein/E2F pathway only in TRbeta(PV/PV) mice. The liganded TRbeta repressed cyclin D1 expression via tethering to the cyclin D1 promoter through binding to the cyclic AMP response element-binding protein. That repression effect was lost in mutant PV, thereby resulting in constitutive activation of cyclin D1 in TRbeta(PV/PV) mice. The present study revealed a novel molecular mechanism by which an unliganded TRbeta mutant acts to contribute to pituitary tumorigenesis in vivo and provided mechanistic insights into the understanding of pathogenesis of TSH-omas in patients.

Mantle Cell Lymphoma: SAHA Blocks Akt/mTOR Pathway and Reduces Cyclin D1 Protein Levels by Affecting Translation.

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase (HDAC) inhibitor. We have found that SAHA has profound antiproliferative activities against mantle cell lymphoma (MCL) cell lines (ED50 ≅ 1 μM SAHA for 3 days), and this was associated with a rapid decrease of Cyclin D1 protein as seen by western blot. For example, eight hours treatment with SAHA (5 μM), reduced the Cyclin D1 levels by 80 % in three of 3 MCL cell lines (Jeko1, SP49, SP53). In contrast, other HDAC inhibitors including valproic acid and trichostatin A, were unable to reduce protein levels of Cyclin D1 as rapidly or as completely as SAHA in these MCL cell lines. Also, a myelogenous leukemia cell line, K562 expressed low levels of Cyclin D1 and exposure of these cells to SAHA (8 hrs) had almost no effect on protein levels of Cyclin D1, although SAHA had profound antiproliferative effect on these cells. A hallmark of MCL cells is the t(11; 14)(q13; q32) chromosomal change which rearranges the enhancer region of the immunoglobulin heavy chain to the regulatory region of the cyclin D1 gene inducing overexpression of Cyclin D1 mRNA, leading to high expression of Cyclin D1 protein. Levels of Cyclin D1 mRNA (Northern blot) remained elevated in these cell lines during an 8 hr exposure to SAHA (5 μM). Also, stability of Cyclin D1 protein in these MCL lines as measured by pulse-chase assay and cyclohexamide treatment minimally changed during the 8 hours of exposure to SAHA in the MCL cell lines. Taken together, we speculated that translation of the Cyclin D1 may be slowed after treatment of the cells with SAHA. Since translation of Cyclin D1 protein is regulated by eIF4E, the binding activity of eIF4E to the cap site of mRNA was analyzed by immunoprecipitation using 7 methyl-GTP sepharose beads. Binding activity decreased rapidly after the treatment with SAHA. Since hypophosphorylated eIF4EBP binds eIF4E and blocks the function of eIF4E, activity of eIF4E is regulated by eIF4EBP and its phosphorylation status. Furthermore, Akt phosphorylates and activates mTOR which then phosphorylates eIF4EBP and prevents eIF4EBP from binding to eIF4E. We found that eIF4EBP, mTOR, and Akt proteins are markedly phosphorylated resulting in an activated Akt/mTOR pathway in the MCL cell lines. Levels of phospho-eIF4EBP, phosph-mTOR and phospho-Akt decreased rapidly after treatment with SAHA. Also, PI3K inhibitor, LY294002, transiently decreased the level of Cyclin D1 protein. In summary, our data suggest that 1) Akt/mTOR pathway is activated in MCL cells; 2) Translation of Cyclin D1 is dependent on this pathway in these cells; 3) Blockade of this pathway reduces the protein levels of Cyclin D1 rapidly by decreasing its translation; 4) SAHA blocks this pathway through a mechanism other than HDAC inhibition. This discovery may encourage a reappraisal of the mechanism of action of this interesting therapeutic compound and also focus attention to developing additional inhibitors of this pathway in cancers associated with dysregulated Cyclin D1.

Cyclin D1 overexpression in thyroid tumours from a radio-contaminated area and its correlation with Pin1 and aberrant beta-catenin expression.

Cyclin D1 is a target molecule transcriptionally activated by aberrant beta-catenin in Wnt signalling, while prolyl isomerase Pin1 promotes cyclin D1 overexpression directly or through accumulation of beta-catenin in cancer cells. This study aimed to elucidate whether Pin1 was involved in cyclin D1 overexpression and aberrant beta-catenin in thyroid tumourigenesis by examining 14 follicular adenomas (FAa) and 14 papillary thyroid carcinomas (PTCs). All PTCs displayed cyclin D1 overexpression and strong cytoplasmic beta-catenin and/or decreased membrane beta-catenin expression by immunohistochemistry. Overexpression of cyclin D1 mRNA was observed in 45.5% of FAs and 54.5% of PTCs by TaqMan real-time PCR. Pin1 expression was observed in PTC by immunostaining and was confirmed by reverse transcriptase-PCR. There was a strong correlation between cyclin D1 and Pin1/cytoplasmic/membrane beta-catenin expression (p < 0.001), and between Pin1 and cytoplasmic (p < 0.001)/membrane (p = 0.002) beta-catenin expression in thyroid tumours. Mutation of the beta-catenin gene could not be detected in PTC. Western blot analysis demonstrated high levels of cyclin D1 and beta-catenin as well as Pin1 expression in a human PTC cell line possessing wild-type beta-catenin and APC genes. This study suggests that both cyclin D1 overexpression and aberrant beta-catenin expression are of significance in thyroid tumours. Pin1 expression appears to correlate closely with the level of cyclin D1 and aberrant beta-catenin expression in thyroid tumours such as FA and PTC. Pin1 may be an important factor in regulating cyclin D1 and beta-catenin expression during thyroid carcinogenesis.

Real-time quantitative reverse transcription-PCR for cyclin D1 mRNA in blood, marrow, and tissue specimens for diagnosis of mantle cell lymphoma.

Overexpression of cyclin D1 mRNA, found in mantle cell lymphoma (MCL), is a critical diagnostic marker. We investigated the use of real-time reverse transcription-PCR (RT-PCR) for cyclin D1. We studied 97 fresh specimens (50 blood, 30 bone marrow, 15 lymph node, and 2 other samples) from patients diagnosed with a variety of lymphoproliferative diseases, including 25 cases of MCL. We used real-time quantitative RT-PCR to evaluate cyclin D1 mRNA expression. Because blood and marrow specimens may contain only a minority of potentially malignant cells (as opposed to most lymph nodes) and to increase sensitivity, we normalized the cyclin D1 mRNA concentrations to mRNA of a B-cell-specific marker, CD19, as well as to previously characterized beta(2)-microglobulin mRNA. In 16 of 21 cases of MCL with overt disease, the ratio of cyclin D1 mRNA to beta(2)-microglobulin mRNA was increased, but all 21 cases showed increased ratios of cyclin D1 mRNA to CD19 mRNA. Cyclin D1 mRNA was low or undetectable in various lymphoproliferative diseases, including cases of ambiguous immunophenotype. The mRNA ratios were stable over 3-7 days of sample storage. Quantitative RT-PCR for cyclin D1 mRNA normalized to CD19 mRNA can be used in the diagnosis of MCL in blood, marrow, and tissue.

Alteration of cyclin D1 in gastric carcinoma and its clinicopathologic significance

To detect the genetic alteration and abnormal expression of cyclin D1 in gastric carcinoma and investigate its clinicopathologic significance in advanced gastric carcinoma. Proteins of cyclin D1 were detected by immunohistochemistry in 42 cases of advanced gastric carcinoma with their follow-up data available, 27 cases of early stage carcinoma, 21 cases of gastric adenoma, 22 cases of hyperplastic polyp and 20 cases of normal mucosa adjacent to adenocarcinomas. Genetic alteration of cyclin D1 was detected by Southern blot and expression of cyclin D1 mRNA was detected by PT-PCR in 42 cases of advanced gastric carcinoma. Cyclin D1 protein was not expressed in normal mucosa, hyperplastic polyp and gastric adenoma, while it was only positively expressed in gastric carcinoma. The expression rate of cyclin D1 protein in early stage gastric carcinoma, advanced gastric carcinoma and lymph node metastasis was 48.1%, 47.4% and 50.0%, respectively. The amplification of cyclin D1 gene was detected in 16.6% of advanced gastric carcinomas. The overexpression of cyclin D1 mRNA was detected in 40.5% of the samples. There was no significant correlation between cyclin D1 protein expression and age, lymph-node metastasis and histological grading in patients with advanced gastric carcinoma (chi2 = 0.038, 0.059, 0.241, P>0.05). Significant correlation was observed between the expression of cyclin D1 protein and the 5-year survival rate (chi2 = 3.92, P<0.05). Detection of cyclin D1 protein by immunohistochemistry may be useful in the diagnosis of early gastric carcinomas. Patients with positive expression of cyclin D1 protein tend to have a worse prognosis.

Real-time Quantitative RT-PCR of Cyclin D1 mRNA in Mantle Cell Lymphoma: Comparison with FISH and Immunohistochemistry

Presence of the balanced translocation t(11;14)(q13;q32) and the consequent overexpression of cyclin D1 found in mantle cell lymphoma (MCL) has been shown to be of important diagnostic value. Although many molecular and immunohistochemical approaches have been applied to analyze cyclin D1 status, correlative studies to compare different methods for the diagnosis of MCL are lacking. In this study, we examined 39 archived paraffin specimens from patients diagnosed with a variety of lymphoproliferative diseases including nine cases meeting morphologic and immunophenotypic criteria for MCL by: (1) real-time quantitative RT-PCR to evaluate cyclin D1 mRNA expression; (2) dual fluorescence in situ hybridization (FISH) to evaluate the t(11;14) translocation in interphase nuclei; and (3) tissue array immunohistochemistry to evaluate the cyclin D1 protein level. Among the nine cases of possible MCL, seven cases showed overexpression of cyclin D1 mRNA (cyclin D1 positive MCL) and two cases showed no cyclin D1 mRNA increase (cyclin D1 negative "MCL-like"). In six of seven cyclin D1 positive cases, the t(11;14) translocation was demonstrated by FISH analysis; in one case FISH was unsuccessful. Six of the seven cyclin D1 mRNA overexpressing cases showed increased cyclin D1 protein on tissue array immunohistochemistry; one was technically suboptimal. Among the two cyclin D1 negative MCL-like cases, FISH confirmed the absence of the t(11;14) translocation in both cases. All other lymphoproliferative diseases studied were found to have low or no cyclin D1 mRNA expression and were easily distinguishable from the cyclin D1 overexpressing MCLs by all three techniques. In addition, to confirming the need to assess cyclin D1 status, as well as, morphology and immunophenotyping to establish the diagnosis of MCL, this study demonstrates good correlation and comparability between measure of cyclin D1 mRNA, the 11;14 translocation and cyclin D1 protein.

In Situ Hybridization and Reverse Transcription–Polymerase Chain Reaction for Cyclin D1 mRNA in the Diagnosis of Mantle Cell Lymphoma in Paraffin-Embedded Tissues

Mantle cell lymphoma (MCL) is characterized by the chromosomal translocation t(11;14), which involves rearrangement of the bcl-1 proto-oncogene to the immunoglobulin heavy chain gene and results in overexpression of cyclin D1 mRNA. In this study, we evaluated the diagnostic relevance of three methods that may be helpful in the diagnosis of MCL: in situ hybridization (ISH) and a stringent reverse transcriptase–polymerase chain reaction (RT-PCR) protocol for cyclin D1 mRNA, and immunohistochemistry for cyclin D1 protein. The study group included 37 paraffin-embedded specimens (25 from lymph nodes and 12 from extranodal tissues) from 30 patients. MCL diagnosis was performed according to the Revised European-American Classification of Lymphoid Neoplasms. Twenty-nine patients with non-MCL lymphoproliferative disorders comprised the control group. Biotin-labeled ISH was performed in 28 cases of MCL, 24 (86%) of which were found to be positive. As shown by ISH in extranodal tissues, cyclin D1 mRNA was present not only in neoplastic lymphoid cells, but in other cell types as well. For this reason, RT-PCR results were considered reliable for MCL diagnosis only on informative material (from tissues that do not normally express cyclin D1); this method was evaluated as positive in 16 of 18 (89%) MCL cases. Cyclin D1 immunopositivity was present in 20 of 29 (69%) MCL cases. No members of the control group were found to express cyclin D1 mRNA by either ISH or RT-PCR under the stringent conditions used. In conclusion, stringent RT-PCR for cyclin D1 expression can be helpful in MCL diagnosis in paraffin-embedded material from lymph nodes. ISH is a sensitive method for cyclin D1 mRNA detection; its sensitivity is superior to that of cyclin D1 immunohistochemistry and similar to that of the stringent RT-PCR used. ISH is very specific as well, clearly more specific than RT-PCR, because it allows the correlation of molecular findings with morphology. This method can be applied on all types of paraffin-embedded tissues and provides an accurate tool for MCL diagnosis.

Cyclin D1 overexpression in thyroid carcinomas: relation with clinico-pathological parameters, retinoblastoma gene product, and Ki67 labeling index.

Cyclin D1 is a G1 cyclin participating in the control of cell cycle progression through interaction with the retinoblastoma gene product (pRB). The overexpression of positive regulators (such as cyclin D1) has been reported in a variety of neoplasms, but their role in thyroid tumorigenesis is yet to be established. In our series of 54 thyroid carcinomas, cyclin D1 overexpression (detected by both immunohistochemistry and by Northern blotting) was correlated with prognostic variables, proliferative activity and pRB. Cyclin D1 overexpression was observed in 35% of thyroid carcinomas with a significantly higher expression of this cyclin in neoplastic tissues than in matched normal parenchyma. In well-differentiated carcinomas, the cyclin D1 mRNA overexpression was inversely correlated with nodal status (p = 0.03), while the protein product was higher in tumors from patients less than 40 than patients over 40 years of age. Inversely, there was no significant correlation with gender and tumor status, pRB and with proliferative activity.

Blastic Mantle Cell Leukemia: An Unusual Presentation of Blastic Mantle Cell Lymphoma

Six patients had blood and bone marrow manifestations characterized by the presence of morphologically immature or blastic B-lineage lymphoid cells expressing CD5 antigen. The median patient age was 70 years, and the male-to-female ratio was 5:1. The presence or degree of lymphadenopathy and splenomegaly was variable among this group at staging evaluation, although two patients did not have these features. One patient had an antecedent diagnosis of classical nodal mantle cell lymphoma, without prior morphologic blood or bone marrow involvement. Other patients lacked a history of underlying lymphoproliferative disorders. The median white blood cell count was 120 × 109/L. Most patients had thrombocytopenia, whereas only one patient had neutropenia at presentation. Leukemic peripheral blood cells in these six cases were small to medium in size with fine or granular nuclear chromatin and small or inconspicuous nucleoli. The pattern of marrow involvement was interstitial or diffuse, with cells showing immature nuclear features resembling acute leukemia or blastic lymphoma. All tumors demonstrated a consistent immunophenotype of B-cell lineage, surface immunoglobulin positivity, and CD5 antigen expression. The progenitor cell-associated markers CD34 and TdT were not expressed, and CD23 antigen was either negative (three of four cases) or only weakly present (one of four cases). The presence of a karyotypic t(11;14)(q13;q32) was documented in one tumor, whereas two other cases had BCL-1 gene rearrangements by either polymerase chain reaction or Southern blot analysis. Cyclin D1 mRNA overexpression was noted in three of four cases tested. This patient group was characterized by very poor overall survival (median, 3 months; range, 0.5 to 6 months). The aggregate clinical, pathologic, and genetic data in these unusual cases are consistent with de novo or predominant leukemic presentations of blastic mantle cell lymphoma. Accurate diagnosis in such cases is greatly facilitated by cytogenetic studies or the demonstration of BCL-1/cyclin D1 abnormalities.

Cyclin D1 overexpression and p53 mutation status in epithelial ovarian cancer.

To examine the cyclin D1 mRNA expression level in ovarian tumor samples as compared with normal ovaries and to determine the relationship between cyclin D1 overexpression and p53 mutation status in ovarian tumors. mRNA was isolated and cDNA was prepared from 27 epithelial ovarian tumors (3 tumors of low malignant potential (LMP) and 24 cancers) and 6 normal ovaries. The cyclin D1 sequences were amplified by using a thermal cycler in parallel with the beta-tubulin gene as an internal control. The cyclin D1 mRNA expression level relative to beta-tubulin was determined by 32P phosphoimager analysis. To confirm the overexpression of the cyclin D1 protein in ovarian tumor cells, immunostaining was performed. The p53 gene mutation status was examined by direct cDNA sequencing. mRNA levels of cyclin D1 were significantly higher in 21 (78%) of the 27 ovarian tumors than in normal ovaries. Cyclin D1 overexpression was detected in ovarian LMP tumors as well as in ovarian cancer cases. Positive immunostaining of cyclin D1 protein was observed in 10 of 18 (56%) ovarian tumors examined. p53 mutations were found in 11 (61%) of 18 ovarian tumors. Of 11 ovarian tumor cases with p53 mutations, 5 showed overexpression of cyclin D1. All 7 ovarian tumor cases without p53 mutations showed significant cyclin D1 mRNA overexpression. Cyclin D1 overexpression seems to be an early genetic event in ovarian tumor development. Although p53 may be one of the proteins whose function regulates the expression of G1 cyclins, ovarian tumors with no p53 mutation consistently showed cyclin D1 overexpression. Cyclin D1 overexpression may play an important role in the tumorigenesis of epithelial ovarian tumors.

Differential Expression of Cyclin D1 in Mantle Cell Lymphoma and Other Non-Hodgkin's Lymphomas

Mantle-cell lymphomas are associated with a characteristic chromosomal translocation, t(11;14)(q13;q32). This translocation involves rearrangement of the bcl-1 proto-oncogene from chromosome 11 to the immunoglobulin heavy chain gene on chromosome 14, resulting in an overexpression of cyclin D1 mRNA (also known as bcl-1 and PRAD1). In the current study performed on paraffin-embedded tissue, cyclin D1 mRNA could be detected in 23 of 24 mantle-cell lymphomas by reverse transcription polymerase chain reaction (RT-PCR) whereas only 9 of 24 demonstrated a t(11;14) by PCR. However, we also found that cyclin D1 mRNA could be detected in the majority (11 of 17, 65%) of non-mantle-cell lymphomas and in a minority of atypical lymphoid hyperplasias (3 of 7, 43%). Cyclin D1 mRNA expression was not observed in floridly reactive lymph nodes (0 of 9) or in unstimulated lymph nodes (0 of 20), suggesting that it is a sensitive adjunct marker for malignant lymphoproliferative processes, but not specific for mantle-cell lymphoma. A semiquantitative RT-PCR assay was developed that compared the ratio of cyclin D1 to the constitutively expressed gene beta2-microglobulin. Using this assay on a limited number of our specimens, cyclin D1 overexpression in mantle-cell lymphoma could be reliably distinguished from its expression in other non-Hodgkin's lymphomas. This assay for cyclin D1 expression, designed for formalin-fixed, paraffin-embedded tissue, was a very sensitive and specific marker for mantle-cell lymphoma.

EMS1 gene expression in primary breast cancer: relationship to cyclin D1 and oestrogen receptor expression and patient survival.

The EMS1 and CCND1 genes at chromosome 11q13 are amplified in about 15% of primary breast cancers but appear to confer different phenotypes in ER positive and ER negative tumours. Since there are no published data on EMS1 expression in large series of breast cancers we examined the relationship of EMS1 expression with EMS1 gene copy number and expression of mRNAs for cyclin D1 and ER. In a subset of 129 patients, where matched tumour RNA and DNA was available, EMS1 mRNA overexpression was associated predominantly with gene amplification (P = 0.0061), whereas cyclin D1 mRNA overexpression was not (P = 0.3142). In a more extensive series of 351 breast cancers, there was no correlation between cyclin D1 and EMS1 expression in the EMS1 and cyclin D1 overexpressors (P = 0.3503). Although an association between EMS1 mRNA expression and ER positivity was evident (P = 0.0232), when the samples were divided into quartiles of EMS1 or cyclin D1 mRNA expression, the increase in the proportion of ER positive tumours in the ascending EMS1 mRNA quartiles was not statistically significant (P = 0.0951). In marked contrast there was a significant stepwise increase in ER positivity in ascending quartiles of cyclin D1 mRNA (P = 0.030). A potential explanation for this difference was provided by the observation that in ER positive breast cancer cells oestradiol treatment resulted in increased cyclin D1 gene expression but was without effect on EMS1. The relationship between EMS1 expression and clinical outcome was examined in a subset of 234 patients with median follow-up of 74 months. High EMS1 expression was associated with age > 50 years (P = 0.0001), postmenopausal status (P = 0.0008), lymph node negativity (P = 0.019) and an apparent trend for worse prognosis in the ER negative subgroup. These data demonstrate that overexpression of EMS1 mRNA is largely due to EMS1 gene amplification, is independent of cyclin D1 and ER expression and, in contrast to cyclin D1, is not regulated by oestrogen. Independent overexpression of these genes may confer different phenotypes and disease outcomes in breast cancer as has been inferred from recent studies of EMS1 and CCND1 gene amplification.

Mechanism of cyclin D1 (CCND1, PRAD1) overexpression in human cancer cells: analysis of allele-specific expression.

The cyclin D1/CCND1 oncogene (PRAD1) is amplified in 15% of primary human breast cancers and overexpressed in 30-50% of breast cancers, suggesting that mechanisms in addition to DNA amplification may lead to deregulated expression of this gene in breast cancer. Cyclin D1 overexpression at a higher frequency than gene amplification is also seen in a variety of other tumors. Cyclin D1 overexpression without amplification could result from a trans-acting regulatory disturbance or could be a consequence of a clonal regulatory mutation in one allele of the gene. We have, therefore, examined whether the overexpression of cyclin D1 mRNA is derived from one parental allele or both alleles in tumor cell lines with or without amplification of the cyclin D1 gene. Eight tumor cell lines, MCF-7, SK-BR-3, ZR-75-1, U-2-OS, SK-LMS-1, DLD1, HCT15, and HT29, out of 20 tumor cells initially examined were found to be heterozygous at the polymorphic NciI site within exon 4 of the cyclin D1 gene. Polymerase chain reaction and NciI digestion (PCR-RFLP) analysis of genomic DNA demonstrated DNA amplification of one allele in the ZR-75-1 cells and HT29 cells and no such imbalance in cyclin D1 gene copy number in the other cells, consistent with Southern blot analyses. Reverse-transcription polymerase chain reaction analysis and NciI digestion (RT-PCR-RFLP) of total cDNA revealed that the overexpressed cyclin D1 mRNA is preferentially derived from the amplified allele in the ZR-75-1 and HT29 cells. In contrast, the other tumor cells overexpressed cyclin D1 mRNA equally from both alleles. This finding strongly suggests that, in breast, sarcoma, and in colon cancer cells with cyclin D1 overexpression and normal gene copy number, elevated levels of cyclin D1 mRNA result from a trans-acting influence on both alleles rather than a clonal somatic mutation or rearrangement in or near a single cyclin D1 gene.

Amplification and overexpression of cyclin D1 in aggressive human esophageal cancer.

Cyclin D1 is a cell-cycle regulator essential for G1 phase progression and a candidate proto-oncogene implicated in pathogenesis of several human tumor types including esophageal cancers. Association between cyclin D1 gene amplification and clinical outcome was examined using southern blotting in 45 patients with primary esophageal cancer. In cases showing gene amplification, expression levels of mRNA and gene product were further examined by northern blotting, western blotting and immunohistochemical analyses. Amplification of cyclin D1 gene was found in 14 of 45 patients (31. 1%). There was no association between gene amplification and clinicopathological parameters but the frequency of hematogenous recurrence in cases with gene amplification was significantly higher than that in cases without amplification in 30 patients undergoing curative surgery (70.0% vs 25.0%, p<0.05). Furthermore, cumulative survival rate of cases with gene amplification was significantly lower than that of cases without amplification (p<0.05). Overexpression of cyclin D1 mRNA was observed in 12 of 14 cases (85. 7%) showing gene amplification but overexpression of its protein was found in only four cases (28.6%). Amplification of the cyclin D1 gene is a reliable prognostic factor and post-translational modification of this gene may play a functionally important role in development of primary esophageal cancer.

Quantitative analysis of cyclin D1 messenger RNA expression in head and neck squamous cell carcinomas.

Cyclin Dl is thought to play a critical role in the G1/S phase transition of the cell cycle. Amplification of this gene has been reported in several types of human neoplasms including breast, lung, esophageal, and head and neck tumors. In this study, we have analyzed the relative level of expression of cyclin D1 messenger RNA (mRNA) in fresh specimens of head and neck squamous cell carcinoma (HNSCC), and investigated the concordance of the overexpression of cyclin D1 mRNA with gene amplification. Levels of cyclin D1 mRNA were analyzed hy a modified method of competitive reverse transcription‐polymerase chain reaction and levels of cyclin D1 gene amplification were evaluated by Southern blot hybridization in a series of 23 matched normal mucosas and HNSCC. Overexpression of cyclin D1 mRNA was observed in 10 of 23 cases (43.5%) of HNSCC, ranging from 2 to 50‐fold higher than the normal control. Twelve of 23 cases could be evaluated by Southern blot hybridization, and gene amplification was found in only 2 of 12 cases (16.7%), These findings suggest that cyclin D1 plays an important role in tumorigenesis of HNSCC, and gene amplification is not one of the major mechanisms for overexpression of cyclin D1.

Cyclin D1 (PRAD1) alternative transcript b: full-length cDNA cloning and expression in breast cancers.

The cyclin D1/PRAD1 protooncogene is a key regulator of the G1 phase of the cell cycle and has been incriminated in the pathogenesis of a variety of primary human tumors. Recently, part of a novel alternatively spliced cyclin D1 transcript, called transcript b, has been identified. This variant transcript showed a failure of splicing at the 3' end of exon 4 and as a result, the expected protein product is altered at its C-terminus. Because of similar transcript sizes, previous Northern analyses would not have been expected to distinguish the two variants, and the relative levels of the two cyclin D1 transcripts in human tumors is unknown. To elucidate the role of cyclin D1 transcript b, we have isolated cDNA clones of this variant transcript from human breast cancer cell lines and report the sequence of the entire coding region of the cDNA. The protein predicted from the cDNA sequence consists of 274 amino acid residues and lacks a PEST sequence in its C-terminus. Examination of the levels of the two alternative cyclin D1 transcripts in primary breast cancers and breast cancer cell lines by Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) assays showed that the variant transcript b is indeed expressed in primary breast cancers and breast cancer cell lines, but the level of transcript b is dramatically lower than that of the originally reported transcript a of the cyclin D1 gene. In breast cancers, oncogenic overexpression of cyclin D1 mRNA appears to consist overwhelmingly of transcript a, and the role of transcript b, if any, in oncogenesis remains to be established. Science Ireland Ltd.