UNIVERSIDAD COMPLUTENSE DE MADRID, (Spain)
FACULTAD DE MEDICINA
New in oncology
A new pathway in the study of carcinogenesis
RIBOGRAMA: Biomolecular concept and its use in clinical oncology and laboratory analyzes A. Ferreira-Alem達o MD PhD (Universidad Complutense de Madrid)
The Cell CELL WALL CYTOPLASM NUCLEOS DNA
RNA
RIBOSOME PROTEIN CHAIN (TUMOR MARKERS)
biomolecular basis of the cellular machinery FREE AND MEMBRANE RIBOSOMES
ROUGH ENDOPLASMIC RETICULUM
RNA
DNA
FREE RIBOSOMES
MEMBRANE RIBOSOMES
CELLULAR NUCLEUS
CYTOPLASM
Free ribosomes are generally assumed to synthesize intracellular proteins (internal economy of the cell), whereas bound ribosomes synthesize proteins intended for secretion (exportation).
free robosomes
Electron microscopy view
HYPERACTIVE CELL
NORMAL CELL
morphologic quantitative aspect
biomolecular basis of the cellular machinery MEMBRANE RIBOSOMES
Schematic three-dimensional rough endoplasmic reticulum, showing the synthesis of proteins destined for export by ribosomes attached to membranes reticulum (signal sequence).
biomolecular basis of the cellular machinery CONTROL OF PROTEIN SYNTHESIS
Upstream
Downstream side
side RIBOSOME
The protein synthetic capacity of a cell is dictated by a number of processes such: • mRNA availability; • efficiency of translation; • availability of translation factors; • number of ribosomes
Tumor Markers
biomolecular basis of the cellular machinery THE RIBOSOME AS THE COMMON ELEMENT IN THE CELLULAR BIOMOLECULAR PROCESS 1. The evidence accumulated to date indicates that overall the protein synthetic capacity is determined by the number of free ribosomes, which function as an informative sensor of the protein synthesis.
2. Increased cell proliferation is a hallmark of agressive malignant neoplasms, which requires a general increase in protein synthesis and a specific increase in the synthesis of replication-promoting proteins. (Tumor Markers)
3. Ribosome synthesis, or biogenesis, is a complex process dependent on the coordinated synthesis of approximately more then 85 ribosomal proteins, four ribosomal RNA (rRNA) and their subsequent processing and assembly into mature ribosomes.
biomolecular basis of the cellular machinery THE RIBOSOME AS THE COMMON ELEMENT IN THE CELLULAR BIOMOLECULAR PROCESS CELL CYCLE FREE RIBOSOMES QUANTITY
MALIGNANT TRANSFORMED CELL
HYPERPLASIA CELL HYPERPLASIA CELL
NORMAL CELL
NORMAL CELL
INTERPHASE
MITOSIS
INTERPHASE
MITOSIS
G1
S
G2
M
G1
S
G2
M
9,3
8,0
2,0
0,7
9,3
8,0
2,0
0,7
Hours
Hours PRO
MET
ANA
TEL
25,2
2,1
2,1
12,6
Minutes
It has been shown in polyoma virus-transformed cells that one of the key mechanisms for the loss of control of protein synthesis is an inability to decrease ribosome number, which correlated with proliferation of transformed cells in fresh media without addition of serum growth factors (Stanners et al., 1979).
biomolecular basis of the cellular machinery
RIBOGRAM CONCEPT
In the decades of 60s and 70s were published studies concerning the accumulation of free ribosomes during chemical induction of neoplastic growth, through initiation by 7,12dimethylbenz(a)anthracene and promotion caused by 12-O-tetradecanoyl-phorbol-13acetate, in the interfollicular areas of the dorsal skin of mice. Accumulation of ribosomes in the process of oncogenesis
biomolecular basis of the cellular machinery
RIBOGRAM CONCEPT
Several lines of evidence, some well established and recent, say emphatically the idea that RNA content is high in cancer cells and genetic events that lead to cancer are often linked directly or indirectly to the ribosome biogenesis.
A perspective on the biology of malignant cells
biomolecular basis of the cellular machinery
RIBOGRAM CONCEPT - From the subjective nature, relatively of the amount of free ribosomes, a diagnosis of malignancy is made just under the idea of the constancy property that consists of an great increase of free ribosomes in cells that are in a process of multiplication without self control, as in the case of malignant cells; - A malignant cell has a very high free ribosomes density that is a fundamental aspect of its phenotype; - So, naturally, to describe the quantities of free ribosomes it is necessary to create a language reproducible, not subjective, with mathematical translation, through a graphic curve that we call RIBOGRAMA.
Biomolecular basis of the cellular machinery
RIBOGRAM CONCEPT
RIBOGRAMA GRAPHIC CURVE
APOPTOSIS
6mth 12mth 18mth 24mth 30mth 36mth 42mth 48mth 54mth 60mth Legend: Mth = Month A, B, C, D = Concentration levels of free ribosomes
Ribosome Quantification
(Mathematic Graphic)
quantitative
CANCER DANGER LEVEL GRADES
HYPERPROLIFERATION CANCER PHENOTYPE
CLINICAL LEVEL ALARM LEVEL INCRESED LEVEL
Mx
NORMAL LEVEL
N Mn
APOPTOSIS TOXIC LEVEL Time
- ASPIRIN AND AINE’S
ACTION OVER THE PROLIFERATION
- SINVASTATIN AND DERIVATIVES - METFORMINE RIBOGRAMA-GRAPHIC™
Latency time (LT)
Monitorization
CLINICAL LEVEL ALARM LEVEL INCRESED LEVEL
Max
NORMAL LEVEL
Min
APOPTOSIS
TL (low level)
tяВоT
T1
T2
TOXIC LEVEL
biomolecular basis of the cellular machinery THE RIBOSOME NUMBER AS A QUANTITATIVE PARAMETER OF THE MALIGNANT PHENOTYPE TREND
1.
The RIBOGRAME graphic curve reflects a dynamic idea of changes in numerical values or quantities of all the free ribosomes.
2.
The density of free ribosomes is the trend of the phenotype of the malignancy of a cell, relatively to a normal range, of a community of cells under observation in regard to biomolecular behavior, representing a malignant growth of the cell phenotype, above a certain level of increase.
3.
It will be very important produce predictive mechanistic models based on tumor dynamics, reflecting the translation in the cell phenotype.
NOVELTY OF THE WORK: The purpose of this research is to present a non-invasive laboratory diagnostic method in the study of the dynamics of the current cancer process, with consequences to the - diagnosis, screening and monitoring, founded on technical and current concepts of Molecular Biology.
This new method allows a diagnosis in a "pre-carcinoma in situ� stage, since it is based on the phenotype of cells, composed of very high quantities of free ribosomes in their cytoplasm, which can be quantified by means of the of the flow cytometry, after its isolation, and labelled with fluorochromes.
The repeated records of those quantities of free ribosomes, establishes a graphic curve (RIBOGRAMA) that represents the degree of a malignant tendency, which above a certain level of concentration, allows to say that the cells in a tissue can be considered in a process of developing cancer, before any macroscopic and microscopic viewing.
CHARACTERISTICS OF MALIGNANCY VERSUS COUNTING FREE RIBOSOMES
 While Ribograma method gives a quantity idea of the cell phenotipic view, the descriptions of Electron Microscope (EM) view of the cells with phenotypic characteristics of malignancy vary by greater or lesser degree of differentiation of its texture or morphology, but these descriptions are subjective, depending on the observer.  In the descriptions of EM, these mechanisms of malignant transformation cannot be evaluated by mathematic criteria, once the described features of the properties of malignant transformed cells, growing in cell culture or in vivo, as observed by techniques of cell biology or molecular biology do not allow a rigorous quantitative assessment, because they depend on subjective criteria, without concrete numbers or standardized numerical limits.
RIBOGRAMA METHOD VERSUS TUMOUR MARKERS The proposed present quantitative method followed the construction and analysis of individual recording of quantities of free ribosomes, which allows to analyze and predict the dynamics of perspective directions and trends of cell groups, after making a graphic curve (RIBOGRAMA). Tumour markers are proteins, which, usually, are produced by the cancer cells, what means that when we start using tumor markers it is already established a state of cancer in the cells under study. With the construction of individual (RIBOGRAMA) curve to quantify the abnormal, enlarged and high quantity of free ribosomes - in the temporal aspect – the time latency of each numerical change and, subsequently, the possible transformation and transition to the phase of clinical manifestations (before the change of blood markers and histological changes …), the normalization process or apoptosis, we are monitoring the behaviour of a future cancer cell.
APLICATIONS OF THE RIBOGRAMA METHOD This research project has strong potential economics in the following fields: 1 - Medical screening of cells anywhere in the body (in the countries/governments benfit)
Endoscopies
YES – if increased RIBOGRAMA curve and NO – if nomarl RIBOGRAMA curve
2 - Diagnosis 3 - Evolution of medical treatments of cancer cells
Potencial Aplications of RIBOGRAMA Method in Personalized Cancer Treatment (by menu)
PERSONALIZED CANCER TREATMENT “BY MENU” CANCER PATIENT
in vitro work MALIGNANT CELL (immunogenic wall proteins)
Tumor Cells (Biopsy)
in vitro work Behavior of RIBOGRAMA Curve
Circulating Tumor Cells – CTC (from peripheric blood)
Choice of Chemotherapy Agents ABCDE
BDE B
Wall AG (WAg) −Killer Cells −Dendritic Cells −B Cells −T Cells
D
Patient Malignant Cells
E Covalent Linkage Ab+ Chemotherapy agents
Fractionation
Separation of wall fragments (AG)
From Bone Marrow pelvis prepare:
B
Antibodies
Information from RIBOGRAMA
DE
chemoimmunoconjugates
CHTA/Ab
Cancer Cell Culture
Every 12h/week
Best efect of chemotherapypatient agetns by RIBOGRAMA curve behavior
APLICATIONS OF THE RIBOGRAMA METHOD This research project has strong potential economics in the following fields: 1 - Medical screening of cells anywhere in the body (in the countries/governments benfit)
Endoscopies
YES – if increased RIBOGRAMA curve and NO – if nomarl RIBOGRAMA curve
2 - Diagnosis 3 - Evolution of medical treatments of cancer cells
Potencial Aplications of RIBOGRAMA Method in Personalized Cancer Treatment (by menu) 4 – Danger or benefits of certain additives in the food industry 5 – AIDS, other infectious diseases, etc.. 6 – Pharmaceutical Industry 7 – Pure and applied research in Biosciences 8 – Public health and food industry 9 – Oncologic and aging research
The method has great medical and financial impact, firstly, by now, in the lowering significantly the morbidity and mortality of the colorectal cancer disease, in the world, and, after, with other organs and systems.
“Raise new questions, explore new possibilities, and regard old problems from a new angle...� [Albert Einstein]