EP1963861A1

EP1963861A1 - Rna helicase as marker for rare tumours

Info

Publication number: EP1963861A1
Application number: EP06829599A
Authority: EP
Inventors: Kathrin Zeller; Sven Laarmann; Karin Mittmann; Christoph Block; Ralf Janknecht
Original assignee: Signalomics GmbH; Mayo Foundation for Medical Education and Research
Current assignee: Signalomics GmbH; Mayo Foundation for Medical Education and Research
Priority date: 2005-12-14
Filing date: 2006-12-14
Publication date: 2008-09-03
Also published as: US20090239935A1; WO2007068478A1; WO2007068478A8

Abstract

Method for diagnosing diseases in a mammal, preferably in a human, wherein a sample from the mammal is investigated with respect to an increased level of RNA helicase and the increased level of RNA helicase indicates the disease. The disease is oesophageal carcinoma, pancreatic carcinoma, gastric carcinoma, hepatocellular carcinoma, hepatoblastoma and cholangiocellular carcinoma.

Description

"RNA helicase as a marker for rare tumors"

background information

RNA helicases constitute a large superfamily of conserved proteins that perform many functions in biochemical processes involving RNA (ribonucleic acid) molecules, such as transcription, splicing, transport, translation, RNA degradation, and ribosome biogenesis , For example, the RNA helicase can cause unwinding of duplex RNA or interruption of RNA-protein interactions. In addition, RNA helicases may also be involved in gene transcription; they have been shown to function as transcriptional cofactors [Abdelhaleem, 2005].

Since the expression of key proteins involved in cell growth regulation, proliferation, differentiation, and cell death is controlled at the translational level and thus with the involvement of RNA helicases [Abdelhaleem, 2004], there is also a correlation between translational dysregulation and carcinogenesis. progression. For example, the RNA helicase Rck / p54 is overexpressed in colorectal carcinomas and adenomas as well as some other tumors and tumor cell lines. Furthermore, the DDX1 RNA helicase gene is often co-amplified with N-myc in retinoblastoma and neuroblastoma, with a correlation of coamplification to poorer prognosis being discussed [Abdelhaleem, 2004].

p68 (DDX5) and p82 (DDX17) as well as its alternative splicing isoform p72 are closely related members of the DEAD-box family of RNA helicases, characterized by a conserved, Asp-Glu-Ala-Asp (DEAD) sequence. containing motif are characterized. The proteins have been shown to have ATPase, RNA-binding and helicase activities. In addition to its role in RNA metabolism, p68 RNA helicase, for example, continues to be involved in the regulation of gene transcription [Abdelhaleem, 2004]. It interacts with the estrogen receptor-α (ER-α, estrogen receptor-α) to stimulate ER-α-dependent transcription. In addition, interactions between p68 and AIB1 (amplified in breast cancer - amplified in breast cancer), a steroid receptor coactivator that is overexpressed in the majority of breast tumors, are found. zick ef al., 1997], and SRA (steroid receptor rNA activator), a cofactor that functions as RNA [Lanz et al., 1999].

It is believed that the p68 RNA helicase, SRA and AIB1, synergistically stimulate ER-α dependent transcription [Watanabe et al., 2001]. In addition, p68 presumably affects transcription by interacting with RNA polymerase II and further stimulating the transcriptional co-activators CBP and p300 [Rossow and Janknecht, 2003].

The homologous proteins CBP and p300 are endowed with acetyltransferase activity and are therefore able to adapt the chromatin structure as well as the function of a variety of different transcription factors [Goodman and Smolik, 2000; Janknecht, 2002].

In Europe, more than two million people suffer from rare diseases, also known as orphan or rare diseases. Rare diseases are diseases in which less than one in 2,000 sufferers, that would be less than 40,000 sufferers in Germany and less than 200,000 sufferers in the United States. In total, there are approximately 6,000-7,000 rare diseases, of which approximately 1,000 are known as rare tumors or tumor subtypes.

Esophageal carcinomas are malignant epithelial tumors of the esophagus, ie the upper gastrointestinal tract between the larynx and the transition into the stomach. These tumors are 96% squamous cell carcinomas and adenocarcinomas. Within the EU, the incidence is between 3-10 / 100,000. In Germany, esophageal carcinoma is one of the rarer tumors with an increasing tendency. The 5-year survival rate in the United States is 14%. The mortality data are in Germany with 4 / 100,000 almost as high as the incidence, so die every year 4000 people in an esophageal carcinoma. Mortality has hardly changed, but there is a dramatic shift in the frequency of the two histological subtypes. The proportion of adenocarcinomas was below 10% in the 1970s, but this tumor entity has now surpassed the number of squamous cell carcinomas in the US since 1990. Comparable developments are also observed at many centers in Europe and Germany.

As a rule, the diagnosis is made histologically directly by esophagoscopy with multiple biopsies taken. Conventional histology with hematoxylin / eosin staining on paraffin sections allows for a distinction between squamous cell and adenocarcinoma as well as very rare small cell carcinoma. Additional staining methods of the oesophageal mucous membrane are helpful in the detection of macroscopically ambiguous early cancers or multicenter carcinomas. Special tumor markers or molecular genetic markers are not yet available for esophageal carcinoma.

A constant increase in the incidence of pancreatic carcinoma, especially in the western world, can be observed. Today, pancreatic carcinoma is the fifth most common cause of tumor-related death. There are approximately 27,000 deaths annually in the US and 50,000 in Europe. Due to the difficult diagnosis, the aggression of the course of the disease and the ultimately unsatisfactory effectiveness of systemic forms of therapy, only 1-5% of all patients with adenocarcinoma of the exocrine pancreas live 5 years after diagnosis. Therefore, the incidence of this malignancy corresponds approximately to the mortality rate. This poor 5-year survival rate is the worst survival of all tumors in the United States and Europe.

If pancreatic carcinoma is suspected, sonographic examination of the gallbladder and pancreas is performed. In order to exclude other diseases, an X-ray examination of the upper gastrointestinal tract or, even better, an esophageal gastroduodenoscopy is performed. Magnetic Resonance Cholangiopancreatography (MRCP) is one of the best non-invasive imaging modalities for imaging pancreatic stenosis and biliary dilation. The best imaging assessment of the pancreas is by endoscopic ultrasonography.

So far, laboratory medical parameters play only a minor role in diagnostics. Certain tumor markers, such as CA19-9, CA125 and CEA, may confirm the suspicion along with the history and clinical picture, but are of no importance in confirming the diagnosis. Molecular genetic diagnostic methods such as e.g. Although the detection of mutated k-ras in the pancreatic secretase has a high sensitivity, but only a moderate specificity, so it is not routinely used to confirm the diagnosis.

The incidence of gastric carcinoma is decreasing worldwide. In 1995, 20,000 people in Germany fell ill with gastric carcinoma. The relative survival rate is 28% (US 22%) in the lower third of the 5-year tumor survival rates. The localization of tumors has also changed in recent years; in the past, these were predominantly in the distal stomach, now increasingly in the proximal stomach and at the gastroesophageal junction. - A -

At the beginning of the diagnosis, a biopsy endoscopy is performed which detects 94% of gastric carcinomas. There are no specific serum tumor markers for gastric carcinoma. Thus, CA72-4 and CEA can only be used in advanced disease for therapy control. No molecular genetic markers are used in the diagnosis of gastric carcinoma.

Hepatocellular carcinoma (HCC) is a very rare tumor of childhood. It occurs in Western Europe and the US with an incidence of 1-5 / 100,000 inhabitants. Primary hepatic tumors account for approximately 1-1.5% of all pediatric neoplasms and have an incidence of 1.5 / 1,000,000 children at the age of 15 years. Hepatoblastoma is the most common primary liver tumor in this age group followed by HCC. This usually occurs in adolescents. In adults, HCC is the most common form of liver cancer. The main symptoms are palpable liver mass, abdominal pain and, in advanced cases, cachexia and jaundice. The serum alpha-fetoprotein is often elevated. In children, HCC can be a complication of viral hepatitis, especially in endemic areas but also as a result of metabolic diseases. HCC in an otherwise healthy liver is more likely to be seen in childhood than in adults. The HCC is extremely chemoresistant, and most of the diagnosis is already advanced. Accordingly, the 5-year survival rate is only about 6%.

In hepatocellular carcinoma, the tumor marker alphai-fetoprotein is known to be a sensitive and specific serum marker for the presence of hepatocellular carcinoma, with hepatitis C and chronic liver diseases also affecting this marker. In combination with the detection of isoenzymes of gammaglutamyltranspeptidase or abnormal plasma prothrombin, the sensitivity can be increased to 78%.

Cholangiocellular carcinoma is fundamentally different from hepatocellular carcinoma in that it does not derive from hepatocytes but from the bile duct epithelium. Pathogenetically, it is more akin to adenocarcinoma than to hepatocellular carcinoma, although mixed tumors are occasionally diagnosed. There is no association with viral hepatitis, liver cirrhosis or hepatic metabolic defects. Cholangiocellular carcinomas are often associated with inflammatory bowel disease and primary sclerosing cholangitis and are prognostically unfavorable.

Mortality for hepatocellular and cholangiocellular carcinoma is very high, with only a 6% 5-year survival rate in the United States. When cholangiocellular Carcinoma so far no molecular genetic markers are used in diagnostics.

It is therefore the object of the present invention to provide a method for the diagnosis and treatment of tumors, in particular of esophageal carcinoma, pancreatic carcinoma, gastric carcinoma, hepatocellular carcinoma and hepatoblastoma and cholangiocellular carcinoma in mammals, especially in humans, to provide.

Summary

The present invention relates to methods and materials for the diagnosis and treatment of tumors, preferably esophageal carcinoma, pancreatic carcinoma, gastric carcinoma, hepatocellular carcinoma as well as hepatoblastoma and cholangiocellular carcinoma in mammals. The invention likewise relates to a method for producing a corresponding diagnostic agent. The invention further identifies molecules that inhibit the activity of RNA helicase (e.g., p68, p72 and / or p82 RNA helicase). The invention further relates to a method of treating cancer (e.g., esophageal carcinoma, pancreatic carcinoma, gastric carcinoma, hepatocellular carcinoma, hepatoblastoma, cholangiocellular carcinoma). The methods and materials provided herein may be used for diagnosis in mammals having esophageal carcinoma or pancreatic carcinoma or gastric carcinoma or hepatocellular carcinoma or hepatoblastoma or cholangiocellular carcinoma. Diagnosing these cancers may allow physicians to treat the mammal earlier than without diagnosis of the mammal. Early onset of appropriate treatment for cancer (e.g., esophageal carcinoma, pancreatic carcinoma, gastric carcinoma, hepatocellular carcinoma, hepatoblastoma, cholangiocellular carcinoma) may give the mammal a better chance of surviving the cancer.

The methods and materials provided herein can also be used to identify molecules that inhibit helicase (e.g., p68, p72 and / or p82 RNA helicase) activity. Such molecules can be used to treat mammals having cancerous cells (eg, esophageal carcinoma, pancreatic carcinoma, gastric carcinoma, hepatocellular carcinoma, hepatoblastoma, or cholangiocellular carcinoma) so as to reduce the number of cancer cells (eg, 10 , 20, 30, 40, 50, 60, 70, 80, 90 or 100 percent reduction).

In general, this document particularly highlights a method to determine if a Mammalian cancer (eg, esophageal carcinoma, pancreatic carcinoma, gastric carcinoma, hepatocellular carcinoma, hepatoblastoma, cholangiocellular carcinoma, but not breast cancer) or not. The method involves determining whether a sample of a mammal has an increased amount of an RNA helicase (eg p68, p72 and / or p82 RNA helicase) polypeptide, wherein the presence of an increased amount indicates that the mammal has cancer (eg Esophageal carcinoma, pancreatic carcinoma, gastric carcinoma, hepatocellular carcinoma, hepatoblastoma, cholangiocellular carcinoma). The mammal may be a human or else another mammal, eg a horse or a pig. The sample may be a tissue sample from the organ to be diagnosed (suspected organ). In the case of esophageal carcinomas, this is a sample from the esophagus, in the case of pancreatic carcinomas a sample from the pancreas, in the case of pancreatic carcinomas, a sample from the pancreas. Carcinoma a sample from the stomach, in hepatocellular carcinoma, hepatoblastoma or cholangiocarcinoma carcinoma each a sample from the liver.

The method of the invention for diagnosing tumors is based on the qualitative and / or quantitative determination of one or more RNA helicases in a tissue sample or in samples derived therefrom (e.g., tissue lysates or the like). These are preferably helicases of the DEAD-box family, which are known to the person skilled in the art. In a particularly preferred embodiment, the amount of at least one of the helicases from the group of p68, p72 and / or p82 RNA helicases is examined. The diagnostic method according to the invention therefore depicts the use of RNA helicases as tumor-associated markers.

The p68 RNA helicase polypeptide assayed according to the invention may have the sequence given in FIG. However, the RNA helicase polypeptide may also be a p72 RNA helicase polypeptide. The p72 RNA helicase polypeptide may have the sequence shown in FIG. The RNA helicase polypeptide may be a p82 RNA helicase polypeptide. The p82 RNA helicase polypeptide may have the sequence as described for p82 in FIG.

Furthermore, this document particularly relates to a method of treating a mammal (eg, a human) with cancer (eg, esophageal carcinoma, pancreatic carcinoma, gastric carcinoma, hepatocellular carcinoma, hepatoblastoma, cholangiocellular carcinoma). The method involves administration of an inhibitor of RNA helicase polypeptide activity in the mammal. Preferably, these are helicases of the DEAD-box family. In a particularly preferred embodiment, an inhibitor from the group of substances is administered, the activity of the At least reduce RNA helicases p68, p72 and / or p82. Advantageously, the treatment according to the invention reduces the growth of the tumor. It is of particular advantage if the treatment reduces the number of cancer cells in patients (mammals) compared to the untreated patient. For the purposes of this invention, however, it is already a treatment success, provided that the tumor shows only a low growth or stagnates in growth

For the purposes of the invention, any substance which leads to a lower activity of the RNA helicases, preferably the p68, the p72 and / or the p82 helicase, in the tumor cell is considered an inhibitor of the RNA helicase. This definition includes inhibitors of gene expression as well as inhibitors of enzymatic activity.

The mammal to be treated can be a human. The inhibitor may e.g. reduce the p68 RNA helicase polypeptide activity in the cancer cells (e.g., from esophageal carcinoma, pancreatic carcinoma, gastric carcinoma, hepatocellular carcinoma, hepatoblastoma, cholangiocellular carcinoma) in the mammal. The inhibitor may e.g. a siRNA molecule, anti-sense oligonucleotide or ribozyme which reduces the expression level of the p68 RNA helicase polypeptide in cancer cells (eg esophageal carcinoma, pancreatic carcinoma, gastric carcinoma, hepatocellular carcinoma, hepatoblastoma, cholangiocarcinoma carcinoma) ,

The inhibitor employable in the present invention may also be an inhibitor of p72 RNA helicase polypeptide activity in cancer cells (e.g., esophageal carcinoma, pancreatic carcinoma, gastric carcinoma, hepatocellular carcinoma, hepatoblastoma, cholangiocarcinoma carcinoma). The inhibitor may e.g. be an siRNA molecule, anti-sense oligonucleotide or ribozyme that reduce the expression level of p72 RNA helicase polypeptide in cancer cells (eg esophageal carcinoma, pancreatic carcinoma, gastric carcinoma, hepatocellular carcinoma, hepatoblastoma, cholangiocarcinoma carcinoma) can.

However, the inhibitor may also reduce the p82 RNA helicase polypeptide activity in the cancer cells (eg, from esophageal carcinoma, pancreatic carcinoma, gastric carcinoma, hepatocellular carcinoma, hepatoblastoma, cholangiocarcinoma carcinoma) in the mammal. The inhibitor may be, for example, an siRNA molecule, antisense oligonucleotide or ribozyme which regulates the expression level of p82 RNA helicase polypeptide in cancer cells (eg esophageal carcinoma, pancreatic carcinoma, gastric carcinoma, hepatocellular carcinoma, hepatoblastoma, cholangiocarcinoma carcinoma ) can reduce. In a particularly advantageous embodiment of the invention, at least two inhibitors may also be administered in a therapeutically effective amount. Particularly advantageous is a combination of an inhibitor of p68 RNA helicase with an inhibitor of p72 RNA helicase or p82 RNA helicase. The inhibitors may be present in equal or different proportions in the pharmaceutical composition.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, or other references referenced herein are incorporated by reference for their entirety.

The exemplary embodiments specified here serve only to illustrate the invention and are not intended to be limiting.

Other features and advantages of this invention will be apparent from the following detailed description and from the claims.

Description of the figures

Fig. 1

Characterization of the anti-p68 (555-576) antibody. (A) Anti-p68 western blot of whole cell lysates from the human breast cancer cell lines MDA-MB-231 and Hs578T. (B) Peptide competitions. A peptide comprising amino acids 555 to 576 of the p68 RNA helicase suppresses the recognition of endogenous p68 RNA helicase by the anti-p68 (555-576) antibody in a Western blot with Hs578T cell extracts, whereas an unrelated peptide does not. (C) p68 RNA helicase expression in MDA-MB-231 or Hs578T breast cancer cells was detected by immunostaining using the anti-p68 (555-576) antibody. DNA was stained with a Hoechst dye.

Fig. 2

Immunohistochemical analysis of p68 expression (white, fluorescence signal) in liver tissue using the α-p68 (555-576) antibody. (A) normal tissue. (B, C) Hepatocellular carcinoma. Fig. 3

Immunohistochemical analysis of p68 expression (white, fluorescence signal) in human bile duct tissue using the α-p68 (555-576) antibody. (A) normal tissue (bile duct). (B, C) Cholangiocarcinoma.

Fig. 4

Immunohistochemical analysis of p68 expression (white, fluorescent signal) in pancreatic tissue using the α-p68 (555-576) antibody. (A) normal tissue. (B, C) Ductal adenocarcinoma.

Fig. 5

Immunohistochemical analysis of p68 expression (white, fluorescent signal) in gastric tissue using the α-p68 (555-576) antibody. (A) normal tissue. (B, C) Adenocarcinoma.

Fig. 6

Immunohistochemical analysis of p68 expression (white, fluorescence signal) in esophageal tissue using the α-p68 (555-576) antibody. (A) normal tissue. (B) adenocarcinoma.

Fig. 7

Nucleic acid sequence encoding human p68 RNA helicase polypeptide (NM_004396, coding region 171-2015).

Fig. 8

Amino acid sequence of the human p68 RNA helicase polypeptide (NP_004387).

FIG. 9 Nucleic acid sequence coding for human p82 RNA helicase polypeptide or for p72 isoform (NM_006386, coding region for p82: 75-2264, coding region for p72: 312-2264).

Fig. 10

Amino acid sequence of the human p72 or p82 RNA helicase polypeptide (the amino acid sequence for p82 starts with amino acid 1 and ends with amino acid 729, the amino acid sequence for p72 starts at amino acid residue 80 and ends with amino acid 729 (NP_006377).

Fig. 11

(A) Human RKO colon cancer cells were infected three times with a mixture of 2 retroviruses (right lane) and control viruses (left lane). Retrovirus 1 encodes the expression of a shRNA

(small-hairpin RNA) directed against human p68, retrovirus 2 a shRNA directed against human p72. After 48 h, protein extracts were prepared from the cells and separated by gel electrophoresis. In the illustrated Western blots for these protein extracts, there is a marked down-regulation of p68 and p72 / p82 protein, whereas the control protein actin is not affected. Cyclin D1 and c-Myc were also down-regulated in the presence of the p68 + p72 / p82 shRNAs. In addition, an up-regulation of the activated form of caspase 3 can be seen.

(B) RKO cells as in A) were seeded at 25% confluency and examined 2 days later under a phase contrast microscope. The control cells form a nearly confluent cell lawn, whereas the cells infected with p68 + p72 / p82 shRNA scarcely proliferated and rounded off in large numbers (bright in the picture).

(C) and (D) 3.5x10 ⁶ RKO cells treated as described under A) were injected into the left flank of nude mice. After 32 days, resulting tumors were isolated. (C) representatively shows one tumor of control and p68 + p72 / p82 shRNA treated cells. (D) shows the statistical evaluation of the experiment with respect to the examined tumor mass with 8 mice each in both groups. The average tumor mass for the cells treated with p68 + p72 / p82 shRNA is markedly reduced compared to that in the control group.

Fig. 12

Immunoblot analysis of p68 RNA helicase expression level in human carcinoma cell lines (A) Hepatocellular carcinoma human Hep-G2 cell cells were lysed, and a normalized amount of lysate was resolved by SDS-Palyacrylamigel electrophoresis (SDS-PAGE) blotted an ECL Western blot membrane. The detection was carried out with the anti-p68 antibody α-p68-1583 primary antibody followed by a peroxidase-coupled secondary antibody and subsequent ECL reaction. The increased expression of p68 is determined by comparison with the non-tumorigenic permanent human endothelial cell line Hs BST (see Figure 12 G). (B) Cells of human cell line of pancreatic carcinoma ASPC1 were treated as in (A). The increased expression of p68 is detected by comparison with 12G.

(C) Human cell line PaTu cells of pancreatic carcinoma were treated as in (A). The increased expression of p68 is detected by comparison with 12G.

(D) Cells of human cell line KYSE 70 of poorly differentiated esophageal carcinoma were treated as in (A). The increased expression of p68 is detected by comparison with 12G.

(E) Cells of human cell line KYSE 140 of moderately differentiated invasive esophageal carcinoma were treated as in (A). The increased expression of p68 is detected by comparison with 12G.

(F) Cells of human cell line KYSE 510 of well-differentiated esophageal carcinoma were treated as in (A). The increased expression of p68 is detected by comparison with 12G.

(G) Cells of the non-tumorigenic endothelial cell line Hs BST were treated as in (A). The detection of p68 shows only weak expression.

(H) Mammary carcinoma cell line MCF7 cells were treated as positive control as in (A). The increased expression of p68 is determined by comparison with 12 G and is in agreement with Janknecht, 2006 (WO 2006/002053 A2

Fig. 13

Immunocytochemical detection of increased expression of p68 in human tumor cell lines

(A) Cells of the human cell line Hep-G2 of hepatocellular carcinoma were fixed and visualized after saturation of the unspecific protein binding sites with BSA first with the primary antibody α-p68-1583 and then with a Alexa488 fluorescently labeled secondary antibody in the fluorescence microscope. An increased p68 expression was determined by comparison with the positive control of the human breast cancer cell line MCF7, which was overexpressing as p68.

(B) Cells of the human cell line ASPC1 of the pancreatic carcinoma were treated as in (A) and the overexpression of p68 was determined analogously. (C) Cells of the human cell line PaTu of the pancreatic carcinoma were treated as in (A) and the overexpression of p68 was determined analogously.

(D) Cells of the human cell line KYSE 70 of the weakly differentiated esophageal carcinoma were treated as in (A) and the overexpression of p68 was determined analogously.

(E) Cells of the human cell line KYSE 140 of the moderately differentiated invasively growing esophageal carcinoma were treated as in (A) and the overexpression of p68 was determined analogously.

(F) Cells of the human cell line KYSE 510 of the well-differentiated esophageal carcinoma were treated as in (A) and the overexpression of p68 was determined analogously.

(H) Mammary carcinoma cell line MCF7 cells were treated as positive control as in (A). The increased expression of p68 is determined by comparison with 12 G and is in agreement with Janknecht, 2006 (WO 2006/002053 A2).

Detailed description

The present invention includes methods and material for targeting rarer cancers such as hepatoblastoma, hepatocellular, cholangiocarcinoma, pancreatic, esophageal, and gastric cancers in mammals (eg, humans, dogs, cats, horses, cattle, goats, pigs, and rodents). to diagnose and treat.

According to the invention, known methods are used for the purpose of diagnosis for the quantitative and qualitative determination of an RNA helicase in the affected tumor tissues. In particular, methods / methods are used to determine if a patient sample contains an elevated RNA helicase (e.g., p68, p72, and / or p82 RNA helicase) polypeptide levels. As indicated herein, the patient may be classified as "cancerous" or at least "suspected of being cancerous" if the level of RNA helicase is e.g. a RNA helicase of the DEAD-box family such as p68, p72 and / or p82 RNA helicase polypeptide in a sample has an elevated level.

The level p68, p72 and / or p82 RNA helicase polypeptide can be determined by measuring each p68, p72, p82 RNA helicase polypeptide, including but not limited to native and mutant to restrict p68, p72, p82 RNA helicase polypeptides. Examples of p68 RNA helicase polypeptides include, without limiting to this, human p68 RNA helicase polypeptides (for example, GenBank ^® Accession No. NP_004387, Fig. 8), horse-p68 RNA helicase polypeptides rabbit p68 RNA helicase polypeptides and mouse p68 RNA helicase polypeptides. Examples of p72 RNA helicase polypeptides include, without limiting to this, human p72 RNA helicase polypeptides (for example, GenBank ^® Accession No. NP_006377, Fig. 10), horse-p72 RNA helicase polypeptides rabbit p72 RNA helicase polypeptides and mouse -p72 RNA helicase polypeptides.

Examples of p82 RNA helicase polypeptides include, without limiting to this, human p82 RNA helicase polypeptides (for example, GenBank ^® Accession No. NP_006377 Fig. 10), horse-p82 RNA helicase polypeptides rabbit p82 RNA helicase polypeptides and mouse p82 RNA helicase polypeptides.

The term "elevated level" as used herein refers to a level of RNA helicase (eg p68, p72 and / or p82 RNA helicase) polypeptide that is higher than a reference level for an RNA helicase (eg p68, p72 and / or p82 RNA helicase) polypeptide The term "reference level" is used herein to refer to an RNA helicase (p68, p72 and / or p82 RNA helicase) polypeptide level, as is typical of non-cancerous / healthy mammals (eg people) is expressed. The reference level of a p68 RNA helicase polypeptide may be, for example, the average level of p68 RNA helicase polypeptide contained in samples from 50 randomly selected healthy mammals of the respective species.

It is advantageous if comparable samples are used to determine if the considered level is an elevated level or not. For example, the average p68, p72 and / or p82 RNA helicase polypeptide level in healthy normal tissue samples of a particular organ from the same randomly selected group of the same mammal species may be a certain number X units / g normal tissue, while the average corresponding level of p68, p72 and / or p82 RNA helicase polypeptide in the tumor tissue of the same organ from the same randomly selected group mammals thereof, namely, for example, is a smaller amount Y units / g tumor tissue.

The reference level for the RNA helicase polypeptide level in the tumor tissue sample from a particular organ would thus be the level of RNA helicase polypeptide in the normal tissue sample of the corresponding organ. For tumor samples taken from another tissue If this were done, the determination of whether or not its RNA helicase polypeptide level is elevated would, if possible, be made by reference to the level of RNA helicase polypeptide in the associated normal tissue. Belonging in this context refers to the fact that the normal tissue was taken from the same organ (but possibly from another patient), from which also the tumor originates; The determination of the reference level would then take place from this normal tissue. Accordingly, the reference level may be the result of a determination from the non-diseased tissue parts of the identical organ or else from the corresponding non-diseased tissue of another patient. In both cases, it is a reference sample to be used for the diagnosis.

However, at least the determination of RNA helicase polypeptide levels in a tumor sample must be based on a reference level determined in normal tissue that belongs to the same tissue type as the tumor sample, even if the normal tissue sample is from another individual. For example, a liver tumor sample should be compared to a normal liver tissue sample from the same patient; if this is not possible, a comparison with a normal tissue sample from the liver of another patient should alternatively be used.

The level indicative of the pathological condition has no absolute value. Rather, the increased level for an RNA helicase (e.g., p68, p72, or p82 RNA helicase) polypeptide may basically assume any value, provided that the level is higher than the corresponding reference level for the RNA helicase (Ex p68, p72 and / or p82 RNA helicase) polypeptide. For example, an elevated level for an RNA helicase polypeptide may be 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20-fold, or even more, several times higher than the reference level for an RNA Helicase polypeptide in the reference sample. Furthermore, a reference level may represent any value, it may also be 0, for example, for the p68 RNA helicase polypeptide. In this case, each level greater than 0 would be an elevated level for the p68 RNA helicase polypeptide.

In essence, any known method can be used to determine the level of an RNA helicase (e.g., p68, p72 and / or p82 RNA helicase) polypeptide present in a sample. For example, For example, anti-p68 RNA helicase polypeptide antibodies can be used to examine the level of expression of the p68 RNA helicase polypeptide in a sample.

In some embodiments, the level of a p68, p72 and / or p82 RNA helicase polypeptide in a sample can be determined using protein chemical detection methods such as Western blots or immunochemical techniques. Another method that may be used to study a p68, p72 and / or p82 RNA helicase polypeptide in a sample may be functional. For example, an ATPase assay can be used to determine whether or not a tissue sample contains an elevated level of p68 RNA helicase polypeptide.

The level of an RNA helicase (eg, p68, p72, p82 RNA helicase) polypeptide present in a sample can also be determined by measuring a level of mRNA containing an RNA helicase (eg, p68, p72, p82 RNA helicase ) Polypeptide encoded. Any method can be used to measure the level of RNA encoding an RNA helicase (e.g., p68, p72, p82 RNA helicase) polypeptide, including, but not limited to, PCR-based methods. For example, For example, RT-PCR can be used with oligonucleotide primers designed to amplify nucleic acid (e.g., RNA) encoding a p68, p72, and / or p82 RNA helicase polypeptide.

Any methods can be used to identify primers that are capable of amplifying nucleic acid or nucleic acids encoding an RNA helicase polypeptide. For example, a computer algorithm can be used for nucleic acid searching a database (for example, GenBank ^®) to p68 RNA helicase.

Any methods can be used to analyze the amplified products. For example, amplified products corresponding to p68, p72 and / or p82 RNA helicase mRNA can be fractionated by gel electrophoresis and the level of the p68, p72 and / or p82 RNA helicase-specific product determined by densitometry. Alternatively, the level of the p68, p72 and / or p82 RNA helicase-specific product can be determined via quantitative RT-PCR using fluorescent signal molecules or dyes.

Any type of sample, including a tissue sample, can be used to determine the level of an RNA helicase (p68, p72 and / or p82 RNA helicase) polypeptide. Furthermore, any method can be used to obtain a sample. For example, a tissue sample can be obtained via a biopsy. Once received, a sample can be processed prior to measuring the level of an RNA helicase (p68, p72 and / or p82 RNA helicase) polypeptide. For example, a tissue sample can be treated to obtain mRNA. Once obtained, the mRNA can be evaluated to determine the level of p68, p72 and / or p82 RNA helicase mRNA present. In another example, a tissue sample may be processed to obtain cell lysate. Is this cell lysate once available for use? In addition, it may be analyzed using anti-RNA helicase polypeptide antibodies (eg, p68, p72, and / or p82 RNA helicase polypepetide antibodies) to increase the level of RNA helicase polypeptide (eg, p68, p72, and / or p82 RNA helicase polypeptide) in the sample.

The present invention provides a method to aid medical or research professionals in determining whether or not a mammal has cancer. Healthcare professionals may be, for example, doctors, nurses, medical-technical laboratory personnel or pharmacists. Research personnel may include, for example, working group leaders, technical assistants, PhDs and doctoral candidates.

The diagnostic methods according to the invention can advantageously be carried out with an antibody directed against the RNA helicase polypeptide, preferably against the p68 RNA helicase polypeptide whose binding via an immunological method z. B. an immunohistochemical staining or immunoblot is detected.

p68 as a therapeutic target

The present document provides a method of treating mammals, especially humans, who have cancer (eg, mammals having a hepatoblastoma, a hepatocellular, a cholangiocarcinoma, a pancreatic, a gastric, or an esophageal carcinoma ). The method of the invention may e.g. be used to treat cancerous mammals in a manner that reduces the number of cancer cells (eg, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% reduction) or But the growth of the tumor slows down or even stagnates. The method according to the invention is basically applicable to all types of tumors. However, it is not claimed for breast cancer.

According to the method of the invention, at least one compound is administered to a cancer patient, which compound reduces the level of RNA helicase (e.g., p68, p72 and / or p82 RNA helicase) polypeptide activity in the mammalian cancer cells. Any type of compound can be used to reduce the level of RNA helicase (e.g., p68, p72 and / or p82 RNA helicase) polypeptide activity in the mammalian cancer cells.

It can e.g. any inhibitor of p68, p72 and / or p82 RNA helicase polypeptide activity (e.g.

ATPase inhibitors). In a preferred embodiment, antisense nucleic acid molecules, shRNA (small hairpin RNA), siRNA molecules, RNAi constructs and / or PNA oligos can be used to reduce the level of p68, p72 and / or p82 RNA helicase polypeptide activity in a mammalian cancer cells by adjusting the level of p68, p72 and / or p82 RNA helicase) polypeptide expression in the cells is reduced.

In some cases, one or more compounds may be administered to a cancerous mammal such that the level of more than one RNA helicase polypeptide activity is reduced in mammalian cancer cells. For example, a single compound can be administered to reduce the level of p68 and p72 RNA helicase polypeptide activity. In another example, two compounds may be administered to a cancer patient: a compound (eg, a siRNA molecule that is directed against expression of the p68 RNA helicase polypeptide) that can reduce the level of p68 RNA helicase polypeptide activity, and another Compound (eg, an siRNA molecule that is directed against both the expression of p72 and p82 RNA helicase polypeptides) that can reduce both the level of p72 and p82 RNA helicase polypeptide activity.

The invention will be described in more detail in the following embodiments; but these do not limit the scope of the invention, as described in the claims, a.

Exemplary embodiments

1. Immunohistochemical detection of p68 RNA helicase

A rabbit polyclonal anti-p68 antibody was developed to stain tissue samples immunohistochemically. This is directed against amino acids 555 to 576 and was affinity purified against the corresponding oligopeptide (α-p68- (555-576)). This antibody specifically recognizes endogenous p68 RNA helicase (see Fig. 1) [Rossow and Janknecht, 2003] and was used in the present example for immunohistochemical staining of paraffin-embedded tissue samples. Similarly, two additional antibodies were obtained from rabbits, one of which, α-p68-1583, is directed against amino acids 501 to 524 and the other, α-p68-1581, against amino acids 4 to 25.

For immunohistochemical staining, tissue microarrays were used which, in addition to normal tissue samples, comprise numerous tumor samples of a particular tumor form, the normal and abnormal tissue samples being in part from the same patient. After deparaffinization and rehydration of the approximately 10 μm thick tissue sections, followed by heat-induced antigen retrieval, nonspecific protein binding sites were saturated with a BSA (bovine serum albumin, bovine serum albumin) solution. p68 was detected in the tissues using the anti-p68 primary antibody α-p68- (555-576) and an AlexaFluor488 secondary antibody conjugate (goat), and after being embedded in Moll / Dabco using a fluorescence microscope (Axiovert 200, Zeiss). An alignment of the fluorescence signals with previously documented autofluorescence or unspecific background binding of the secondary antibody enables a precise determination of the specific p68 RNA helicase signal. Even slight differences in expression can be represented by this methodology by a good signal-to-background ratio.

In addition, a further evaluation of the results with the antibody α-p68-1583 confirmed the statements made with the antibody α-p68- (555-576). For further investigations, the anti-p68 antibody α-p68-1581 can also be used.

We examined tissue microarrays with samples from hepatocellular carcinomas, cholangiocarcinomas, pancreatic carcinomas, gastric cancer and esophageal carcinomas.

1.1 Bearings explanation for the evaluation negative: A sample is classified as "negative" if the localization and intensity of the p68 signal after immunofluorescence staining corresponds approximately to the level of autofluorescence or the unspecific background.

Moderate: Moderately stained refers to samples showing a mild to moderate increase in the p68 signal compared to autofluorescence and / or the nonspecific background

strong: A sample is classified as "strong" if a significant increase in p68 signal to autofluorescence and / or nonspecific background staining in neoplastic tissue is to be detected, even if the signal is limited to individual structures / cell groups within the tissue sample If necessary, non-neoplastic samples having a corresponding level of expression will also be given this rating.

very strong: This classification is used for samples with an extremely conspicuous and signal-intensive staining pattern. 1.2 Heoatocellular carcinoma Microarrav p68 was stained as described in the hepatocellular carcinoma microarray and the expression levels of the normal and tumor tissues were compared and evaluated. Even before the specific immunohistochemical staining, a high degree of autofluorescence was detected. However, a detected p68 signal was clearly distinguishable from this (FIG. 2).

As can be seen from Table 1, the p68 signal was classified as strong in 37% of the tumor tissue samples in comparison with the autofluorescence and the normal tissue, which corresponds to a significantly increased expression level of the protein. In 9% ("negative") of the cases no or only a very weak expression of p68 in the tissue was detected after comparison with autofluorescence and unspecific background, in 54% the increase of the p68-level was moderate ("moderate"). ).

Table 1 Evaluation of hepatocellular carcinoma Microarray p68 signal for neoplastic samples / total neopl. Samples negative (0) 3/35 (9%)

moderate (1) 19/35 (54%)

strong (2) 13/35 (37%)

very strong (3) 0

1.3 Cholanaio Carcinoma Microarrav

The p68 RNA helicase was stained as described in the Cholangio carcinoma microarray and the expression levels of the normal and tumor tissues were compared and evaluated. Even before the specific immunohistochemical staining, a high degree of autofluorescence was detected. However, a detected p68 signal was clearly distinguishable (FIG. 3).

As can be seen from Table 2, the p68 signal was classified as strong or very strong in comparison with the autofluorescence and the normal tissue in 37% of the tumor tissue samples, which corresponds to a greatly increased expression level of the protein. In 11% ("negative") of the cases no or only a very weak expression of p68 in the tissue was detected after comparison with autofluorescence and unspecific background, in 52% the increase of the p68-level was moderate ("moderate"). ).

Table 2 Evaluation of cholangiocarcinoma Microarray p68 signal for neoplastic samples / total neopl. Samples negative (0) 5/46 (11%)

moderate (1) 24/46 (52%)

strong (2) 16/46 (35%)

very strong (3) 1/46 (2%)

1.4 Pancreatic carcinoma Microarrav p68 was stained as described in the pancreatic tissue samples and the expression levels of the normal and tumor tissues were compared and evaluated. Already before the specific immunohistochemical staining a weak autofluorescence was shown. A detected p68 signal was clearly distinguishable from this (FIG. 4).

As can be seen from Table 3, the p68 signal was classified as strong or very strong in comparison with the autofluorescence and the normal tissue in 48% of the tumor tissue samples, which corresponds to a greatly increased expression level of the protein. In only 3% ("negative") of the cases was there no or only a very weak expression of p68 in the tissue after comparison with autofluorescence. In 49%, the increase of the p68 level was moderate ("moderate").

Table 3 Evaluation of pancreatic carcinoma Microarray p68 signal for neoplastic samples / total neopl. Samples negative (0) 1/33 (3%) moderate (1) 16/33 (49%)

strong (2) 15/33 (45%)

very strong (3) 1/33 (3%)

1.5 Gastric carcinoma Microarray p68 was stained as described in the gastric carcinoma microarray and the expression levels of the normal and tumor tissues were compared and evaluated. Already before the specific immunohistochemical staining a weak autofluorescence was shown. However, a detected p68 signal was clearly distinguishable from this (FIG. 5).

As can be seen from Table 4, the p68 signal was classified as strong in comparison with the autofluorescence and the normal tissue in 24% of the tumor tissue samples, which corresponds to a significantly increased expression level of the protein. In 14% ("negative") of the cases no or only a very weak expression of p68 in the tissue was detected after comparison with autofluorescence and nonspecific background, in 62% the increase of the p68-level was moderate ("moderate"). ). Table 4 Evaluation of gastric carcinoma Microarray p68 signal for neoplastic samples / total number of neopl. Samples negative (O) 7/50 (14%)

moderate (1) 31/50 (62%)

strong (2) 12/50 (24%)

very strong (3) 0

1.6 Ösophaαus carcinoma Microarrav p68 was stained as described in the esophageal carcinoma microarray and the expression levels of the normal and tumor tissues were compared and evaluated. Even before the specific immunohistochemical staining, a high degree of autofluorescence and non-specific binding of the secondary antibody was already evident. However, a detected p68 signal was clearly distinguishable (FIG. 6).

As can be seen from Table 5, the p68 signal was classified as strong in comparison with the autofluorescence and the normal tissue in 25% of the tumor tissue samples, which corresponds to a significantly increased expression level of the protein. In 8% ("negative") of the cases no or only a very weak expression of p68 in the tissue was detected after comparison with autofluorescence and unspecific background, in 67% the increase of the p68-level was moderate ("moderate"). ).

Table 5 Evaluation of esophageal carcinoma Microarray p68 signal for neoplastic samples / total neopl. Samples negative (O) 3/40 (8%)

moderate (1) 27/40 67%)

strong (2) 10/40 (25%)

very strong (3) 0

2. Detection of p68 RNA helicase polypeptide in tissue extracts

The following experiments serve to determine the underlying mechanism of p68 polypeptide overexpression in tumors.

2.1 The expression level of the p68 RNA helicase polypeptide in hepatocellular, cholangiocarcinoma, pancreatic, gastric and esophageal carcinoma is determined by the immunoblot method. For this purpose, the total proteins are isolated from carcinogenic and corresponding normal tissue (non-diseased tissue) of the corresponding types of tumors (commercially available, for example, from BioCat) and separated according to their molecular weight by means of SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The detection of p68 RNA helicase is then carried out in an immunoblot with a specifically directed against the p68 protein polyclonal antibody and a peroxidase-conjugated secondary antibody. The expression level of p68 is then read by the strength of the Enhanced Chemiluminescence System (ECL) and correlated with the amount of p68 protein present in the tissue examined. Comparison with the expression level in normal tissue reveals overexpression in carcinogenic tissue.

2.2

The expression strength of the p68 RNA helicase polypeptide in hepatocellular, cholangiocarcinoma, pancreatic, gastric and esophageal carcinoma is determined by means of an "immunoassay" (eg the ELISA, enzyme linked immunosorbent assay). For this, the entire proteins from carcinogenic and corresponding normal tissue (non-diseased tissue) of the corresponding Tumor species (commercially available, for example, from BioCat) and immobilized in a 'multi-well ELISA plate'. After saturation of protein binding sites with BSA solution, p68 RNA helicase is detected with a polyclonal antibody specifically directed against the p68 protein and with a secondary antibody conjugated with enzyme (eg alkaline phosphatase or peroxidase) or fluorescent dye-conjugated. The expression level of p68 is then read from the strength of the enzymatic detection reaction (a substrate conversion yields a colored product) or fluorescence intensity in an 'LISA reader' and correlated with the amount of p68 protein present in the sample being studied. Comparison with the expression level in normal tissue reveals overexpression in carcinogenic tissue.

These immunoassays are particularly advantageous because they can be automated for high throughput analysis.

2.3 The following experiment aims at the rapid, comprehensive and accurate identification of tumor-specific expression changes of individual genes. Briefly, the Cancer Profiling Array (BD Clonetech) contains an expression profile of 19 tumor forms and corresponding normal tissue, represented by 154 sample pairs, with most tumors represented by at least 10 patient samples. The RNA isolated from the tissue samples was rewritten into cDNA using SMART technology, ensuring the original complexity and relative abundance in the amplified cDNA. The p68 expression is used with a radioactive or alternatively a fluorescently labeled gene-specific DNA probe for hybridization and read the resulting profile with a phosphor imager or a fluorescence scanner. The signal strength correlates with the frequency of the p68-specific cDNA molecules and provides information about the expression strength (mRNA) of the p68 RNA helicase in the original tissue samples.

2.4

Protein stability is largely determined by posttranslational modification. p68 polypeptides are isolated from tumor tissue and corresponding normal tissue with anti-p68 antibodies, and the purified p68 polypeptides are subjected to mass spectrometric analysis. Mass differences may be the exact nature of a post-translational modification that distinguishes the p68 RNA helicase from tumor cells from that from normal cells.

These studies are essential to determine if the promoter of the p68 gene is up-regulated (as in ER alpha) or whether the p68 polypeptide is stabilized by post-translational modifications (eg by blocking ubiquitylation sites by acetylation or sumosylation on the same lysine residues.

Similar experiments are performed to determine the underlying mechanism of p72 and p82 polypeptide overexpression in tumors.

3. p68 expression in the cell line model

3.1

A finding of p68 RNA helicase polypeptide expression level is the investigation of cell extracts in an immunoblot analysis of electrophoretically separated proteins. For this purpose, from a hepatocellular, cholangiopancreatic, pancreatic, gastric or esophageal carcinoma isolated and established cell lines, such as for example, HuCCA-1, Pand, ASPC-1, HUP-T3, NCI-N87, AGS, KYSE, as well as corresponding, non-tumor, but normal tissue isolated, and transformed with the SV40 "large T" antigen cell lines, such as THLE -2 and HeMA ₁ used. These cell lines are cultured under standard cell culture conditions and each considered as a model system for the indicated tumor types.

The expression level of the p68 RNA helicase polypeptide can be determined from total cell lysates or from lysates of the nuclear or cytosolic fraction by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblot with a polyclonal antibody specifically directed against the p68 protein and with a peroxidase antibody. conjugated secondary antibodies are determined. The expression level of p68 is then read by the strength of the detection reaction (e.g., ECL system) and correlated with the amount of p68 protein present in the particular cell line. When normalized to cell number or a reference protein such as e.g. Actin p68 expression levels can thus be compared (see Fig. 12 A to H).

A comparison of the signal strengths between carcinogenic and non-carcinogenic cell line indicates the expression level of the p68 RNA helicase in a tissue sample (compare 12 A to F with negative control 12 G and positive control 12 H).

3.2

Another possibility for determining the expression strength of the p68 RNA helicase polypeptide in the cell lines mentioned under 3.1 is an "immunoassay" (eg an ELISA, enzyme linked For this purpose, the entire proteins from the cultured cell lines are isolated (total cell lysate) and immobilized in a 'multi-well ELISA plate'. After saturation of protein binding sites with BSA solution, p68 RNA helicase is detected with a polyclonal antibody specifically directed against the p68 protein and a secondary antibody conjugated with enzyme (eg alkaline phosphatase or peroxidase) or fluorescent dye-conjugated. The expression strength of p68 is then read from the strength of the enzymatic detection reaction (a substrate conversion leads to a colored product) or the fluorescence intensity in an ΕLISA Reader ¹ and correlated with the amount of p68 protein present in the sample examined. Comparison with the expression level in normal tissue reveals overexpression in carcinogenic tissue.

3.3

In another embodiment, the level of p68 RNA helicase can be detected by means of specific antibodies (such as α-p68- (555-576), α-p68-1582, α-p68-1583) in an immunocytochemical method. For this purpose, cells of the corresponding line are cultivated on glass coverslips and fixed in the subconfluent to confluent state. After permeabilization of the membranes and saturation of nonspecific protein binding sites with a BSA solution, the p68 protein can be isolated by means of the specific binding primary antibodies (α-p68- (555-576), α-p68-1582, α-p68-1583)) and fluorescently labeled secondary antibodies in be made visible in a fluorescence microscope. The fluorescence intensity correlates with the expression strength or frequency of the protein in the cell and can be compared with signals from other cell lines. Furthermore, the subcellular localization (cytosol, nucleus) of the p68 protein can also be determined and give information about its normal or abnormal distribution in the cell (compare 13 A to F).

4. The role of 068 RNA helicase polypeptides in tumor cells

4.1

The following experiments are performed to investigate the influence of p68 RNA helicase polypeptides on the tumor cell phenotype. P68 RNA helicase polypeptide expression is suppressed in human tumor cell lines using RNA interference, and the resulting changes in growth rate and substrate-independent growth are determined. Briefly, siRNA molecules are potent agents to study the effects of downregulation of p68 RNA helicase polypeptide expression in living cells. Especially in the case where p68 is a proto-oncogene its downregulation may inhibit cell transformation.

For example, established tumor cell lines can be transfected with siRNA molecules with the goal of producing RNA interference for p68, and the resulting effect can be assessed via substrate-independent growth (e.g., via the soft agar assay) or cell proliferation.

The following human p68 RNA helicase cDNA sequences can be used as siRNA 'target' sequences:

1. TAAGGAAGATTGTGGATCA

2. TAAGACCTGATAGGCAAAC

3. ACCACAACATTCTTCAGAT

4. ACTTATTCGTCTAATGGAA 5. AGAAGATGTGATGAGCTTA

6. GACAGAGGTTCAGGTCGTT

7. GAACTGCTCGCAGTACCAA

Furthermore, substrate-independent growth in the soft agar assay and colony formation assay can be used to study the ability of p68 polypeptides, the β-catenin mediated transformation of NIH3T3, and To promote RK3E cells (rat). These assays can determine whether p68 RNA helicase polypeptides are involved in the maintenance and / or induction of cell transformation.

Similar techniques can be used to reduce the expression of p72 or p82 RNA helicase. For example, the following human p72 and p82 RNA helicase cDNA sequences can be used as siRNA 'target' sequences:

1. GAGACGCTGTGATGATCTG 2. GATGTCAAGTTTGTGATCA

In vivo experiment

Human RKO colon cancer cells were infected with a mixture of 2 retroviruses. The first retrovirus encodes the expression of a shRNA (small-hairpin RNA) directed against human p68 and the second retrovirus encodes the expression of a shRNA directed against human p72. This shRNA also binds p82. For purposes of linguistic simplicity, therefore, the term "p72 / p82" will be used hereinafter when both p72 and p82 are meant, thus "p72 / p82" is to be understood as "and / or". After infection of the RKO cells three times with this virus mixture (FIG. 11A, right lane) or with control viruses (FIG. 11A, left lane) and subsequent to a 48 h growth phase, protein extracts were prepared using Laemmli buffer. Depicted are corresponding Western blots of these protein extracts. It can be shown that the p68 and p72 / p82 proteins are downregulated more than 80% by shRNAs, whereas the control protein actin is not affected. Interestingly, cyclin D1 and c-Myc were also down-regulated in the presence of the p68 and p72 / p82 shRNAs, which could cause a reduction in cell proliferation. Furthermore, it has been observed that the activated form of caspase 3 has been up-regulated, which may indicate an increased rate of apoptosis. This suggests that overexpression of p68 and / or p72 / p82 may contribute to increased cell proliferation and protection against apoptosis. Both are features that would promote tumor development involving p68 and p72 / p82.

An experiment on cell proliferation also confirmed the observations already described. RKO cells were seeded after infection with the mixture of both retroviruses at 25% confluence and viewed 2 days later in a phase contrast microscope. While control cells proliferated strongly and formed a nearly confluent cell lawn, the cells infected with p68 and p72 / p82 shRNA hardly grew. Furthermore, many round cell bodies (which show a high refractive index in Figure 11 B) could be indicative of dying cells. Obviously, the proliferation of RKO cells is inhibited by p68 and p72 / p82 shRNA.

In an in vivo experiment, the importance of the RNA helicases p68 and p72 / p82 for tumor growth was also demonstrated.

(3.5 x 10 ⁶ ) RKO cells were infected with the mixture of both retroviruses (see above) and subsequently injected into the left trunk side of nude mice. In parallel, the experiment was carried out using control shRNA.

After 32 days, the tumors were isolated and the tumor mass determined. The statistical evaluation of the tumors from 8 mice in each test group clearly shows that the tumor growth is strongly inhibited by p68 and p72 / p82 shRNA (see Fig. 11 C and D).

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Causevic, M., Gordon Hislop, R.G., Kernohan, N.M., Carey, F.A., Kay, R.A., Steele, RJ. , Fuller-Pace, F.V. (2001) Overexpression and poly-ubiquitylation of the DEAD-box RNA helicase p68 in colorectal tumors. Oncogene 20: 7734-43.

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Claims

claims

A method of diagnosing one of the following diseases in a mammal, preferably in a human, characterized in that a sample from the mammal is assayed for an elevated level of RNA helicase, wherein the elevated level of RNA helicase indicates the disease: Esophageal carcinoma, pancreatic carcinoma, gastric carcinoma, hepatocellular carcinoma, hepatoblastoma and cholangiocellular carcinoma.

2. The method according to claim 1, characterized in that the RNA helicase is a DEAD-box helicase

3. The method according to claim 2, characterized in that the RNA helicase is a p68 helicase.

4. The method according to claim 2, characterized in that the RNA helicase is a p 72 helicase.

5. The method according to claim 2, characterized in that the RNA helicase is a p 82 RNA helicase.

6. A method for treating one of the following diseases in a mammal, preferably in a human, characterized in that the mammal is administered an inhibitor of RNA helicase activity: esophageal carcinoma, pancreatic carcinoma, gastric carcinoma, hepatocellular carcinoma, hepatoblastoma and cholangiocellular carcinoma.

7. The method according to claim 6, characterized in that the RNA helicase is a DEAD-box helicase.

8. The method according to claim 6, characterized in that the RNA helicase is a p68 RNA helicase.

9. The method according to claim 6, characterized in that the RNA helicase is a p72 RNA helicase is.

10. The method according to claim 6, characterized in that the RNA helicase is a p82 RNA helicase.

11. The method according to claim 6, characterized in that the inhibitor is a siRNA molecule that reduces the expression of the helicase.